Heterodimeric antibody fc-containing proteins and methods for production thereof

ABSTRACT

Novel heterodimeric antibody-Fc-containing proteins, such as bispecific antibodies, and novel methods for producing such proteins

FIELD OF THE INVENTION

The present invention relates to novel heterodimericantibody-Fc-containing proteins, such as bispecific antibodies, andnovel methods for producing such proteins.

BACKGROUND OF THE INVENTION

Monoclonal antibodies have in recent years become successful therapeuticmolecules, in particular for the treatment of cancer. Unfortunately,however, monoclonal antibodies are often unable to cure diseases whenused as monotherapy. Bispecific antibodies can potentially overcome someof the limitations of monoclonal antibody therapy, e.g. they could beused as mediators to target a drug or toxic compound to target cells, asmediators to retarget effector mechanisms to disease-associated sites oras mediators to increase specificity for tumor cells, for example bybinding to a combination of targets molecules that is exclusively foundon tumor cells.

Different formats and uses of bispecific antibodies have recently beenreviewed by Chames and Baty (2009) Curr Opin Drug Disc Dev 12: 276. Oneof the major obstacles in the development of bispecific antibodies hasbeen the difficulty of producing the material in sufficient quality andquantity by traditional technologies, such as the hybrid hybridoma andchemical conjugation methods (Marvin and Zhu (2005) Acta Pharmacol Sin26:649). Co-expression in a host cell of two antibodies, consisting ofdifferent heavy and light chains, leads to a mixture of possibleantibody products in addition to the desired bispecific antibody.

Several strategies have been described to favor the formation of aheterodimeric, i.e. bispecific, product upon co-expression of differentantibody constructs.

Lindhofer et al. (1995 J Immunol 155:219) have described that fusion ofrat and mouse hydridomas producing different antibodies leads toenrichment of functional bispecific antibodies, because of preferentialspecies-restricted heavy/light chain pairing. Another strategy topromote formation of heterodimers over homodimers is a “knob-into-hole”strategy in which a protuberance is introduced at the interface of afirst heavy-chain polypeptide and a corresponding cavity in theinterface of a second heavy-chain polypeptide, such that theprotuberance can be positioned in the cavity so as to promoteheterodimer formation and hinder homodimer formation. “Protuberances”are constructed by replacing small amino-acid side-chains from theinterface of the first polypeptide with larger side chains. Compensatory“cavities” of identical or similar size to the protuberances are createdin the interface of the second polypeptide by replacing large amino-acidside-chains with smaller ones (U.S. Pat. No. 5,731,168). EP1870459(Chugai) and WO 2009089004 (Amgen) describe other strategies forfavoring heterodimer formation upon co-expression of different antibodydomains in a host cell. In these methods, one or more residues that makeup the CH3-CH3 interface in both CH3 domains are replaced with a chargedamino acid such that homodimer formation is electrostaticallyunfavorable and heterodimerization is electrostatically favorable.WO2007110205 (Merck) describe yet another strategy, wherein differencesbetween IgA and IgG CH3 domains are exploited to promoteheterodimerization.

Dall'acqua et al. (1998 Biochemistry 37:9266) have identified fiveenergetically key amino-acid residues (366, 368, 405, 407 and 409) thatare involved in the CH3-CH3 contact in the interface of a CH3 homodimer.

WO 2008119353 (Genmab) describes an in vitro method for producingbispecific antibodies wherein a bispecific antibody is formed by“Fab-arm” or “half-molecule” exchange (swapping of a heavy chain andattached light chain) between two monospecific IgG4- or IgG4-likeantibodies upon incubation under reducing conditions. This Fab-armexchange reaction is the result of a disulfide-bond isomerizationreaction and dissociation-association of CH3 domains wherein heavy-chaindisulfide bonds in the hinge regions of the parent (originallymonospecific) antibodies are reduced and the resulting free cysteinesform an inter heavy-chain disulfide bond with cysteine residues ofanother parent antibody molecule (originally with a differentspecificity), and simultaneously CH3 domains of the parent antibodiesrelease and reform by dissociation-association. The resulting product isa bispecific antibody having two Fab arms which potentially arecompassed different sequences. It should be noted that the process israndom however and Fab-arm exchange can also occur between two moleculeswith identical sequence or two bispecific molecules can engage inFab-arm exchange to regenerate antibodies comprising the originalmonospecific parent antibody specificity.

It has now surprisingly been found that by introducing asymmetricalmutations in the CH3 regions of the two monospecific starting proteins,the Fab-arm exchange reaction can be forced to become directional andthereby yield highly stable heterodimeric proteins.

SUMMARY OF THE INVENTION

Accordingly, in one aspect, the present invention provides an efficientin vitro method for the production of highly stable heterodimericFc-containing proteins on the basis of stable homodimeric Fc-containingstarting materials. For example, a highly stable bispecific antibody canbe formed with high yield and purity on the basis of two stablemonospecific antibodies as starting material.

Thus, in one aspect, the invention relates to an in vitro method forgenerating a heterodimeric protein, said method comprising the followingsteps:

-   -   a) providing a first homodimeric protein comprising an Fc region        of an immunoglobulin, said Fc region comprising a first CH3        region,    -   b) providing a second homodimeric protein comprising an Fc        region of an immunoglobulin, said Fc region comprising a second        CH3 region,    -   wherein the sequences of said first and second CH3 regions are        different and are such that the heterodimeric interaction        between said first and second CH3 regions is stronger than each        of the homodimeric interactions of said first and second CH3        regions,    -   c) incubating said first protein together with said second        protein under reducing conditions sufficient to allow the        cysteines in the hinge region to undergo disulfide-bond        isomerization, and    -   d) obtaining said heterodimeric protein.

The method can for example be used for in vitro production ofheterodimeric proteins, such as bispecific antibodies, for various uses,such as therapeutic or diagnostic uses. An advantage of this in vitromethod is that heavy-chain/light-chain pairing stays intact during thereaction, so no undesired combinations of heavy chain and light chainsare obtained in the product. This in contrast to some of theco-expression methods described in the prior art (see above) where acommon light chain which can form a functional antibody with both heavychain needs to be found in order to avoid the formation ofnon-functional heavy-chain/light-chain products, because of randomheavy-chain/light-chain pairing in the cell. In addition, the in vitroprocess can be performed in the laboratory which allows greater control,flexibility and yield of the heterodimeric protein than is allowed byco-expression.

The in vitro method of the invention can also be used to create compoundlibraries of larger size, e.g. in a screening method to identifyadvantageous combinations of specificities. For example, for somecombinations of antibody targets, not any bispecific antibody will befunctional, i.e. be able to bind to both targets at the same time andmediate the desired functional effects. In such cases, a bispecificantibody having a desired property, e.g. optimal target binding or cellkilling, may be identified by:

-   -   a) providing a first set of homodimeric antibodies having        different variable regions, wherein said antibodies of said        first set comprise a first CH3 region,    -   b) providing a second set of homodimeric antibodies having        different variable regions, wherein said antibodies of said        second set comprise a second CH3 region,    -   wherein the sequences of said first and second CH3 regions are        different and are such that the heterodimeric interaction        between said first and second CH3 regions is stronger than each        of the homodimeric interactions of said first and second CH3        regions,    -   c) incubating combinations of antibodies of said first set and        of said second set under reducing conditions sufficient to allow        the cysteines in the hinge region to undergo disulfide-bond        isomerization, thus generating a set of bispecific antibodies,    -   d) optionally restoring the conditions to non-reducing,    -   e) assaying the resulting set of bispecific antibodies for a        given desired property, and    -   f) selecting a bispecific antibody having the desired property.

In further aspects, the present invention relates to heterodimericproteins obtained or obtainable by the method of the invention and tomethods for producing heterodimeric proteins of the invention byco-expression in a suitable host cell.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Generation of bispecific antibodies by interspecies Fab-armexchange. The generation of bispecific antibodies after GSH-induced invitro Fab-arm exchange between the indicated EGFR (2F8) and CD20 (7D8)IgG4 antibodies was determined by an ELISA. A concentration series(total antibody) of 0-1 μg/mL was analyzed in the ELISA. Bispecificbinding was higher after Fab-arm exchange between rhesus (Rh) and human(Hu) IgG4 antibodies than between two antibodies of the same species.

FIG. 2: Alignment of the amino acid sequences of the core hinge (i.e.the CPPC sequence in human IgG1 which includes the two cysteine residuesthat potentially form the interheavy chain disulphide bonds andcorresponding residues in other human isotypes or other species) andCH3-CH3 interface of the human and rhesus antibody isotypes.

FIG. 3: Generation of bispecific antibodies using mutant human IgG1engaged for Fab-arm exchange. The generation of bispecific antibodiesafter GSH-induced in vitro Fab-arm exchange between human CD20 (7D8)IgG4 antibody and the indicated human EGFR (2F8) IgG1 antibodies wasdetermined by an ELISA. The presented graph shows average numbers ofthree independent Fab-arm exchange experiments, in which a totalantibody concentration of 1 μg/mL was used for ELISA. Bispecific bindingwas higher after Fab-arm exchange between IgG1-2F8-CPSC-ITL and IgG4-7D8than between two IgG4 antibodies. Combining IgG4-7D8 with eitherIgG1-2F8-CPSC or IgG1-2F8-ITL did not result in bispecific antibodiesunder the conditions used.

FIG. 4: Generation of bispecific antibodies by in vivo Fab-arm exchangeof human IgG4 and mutant IgG1 antibodies. The generation of bispecificantibodies after in vivo Fab-arm exchange in immunodeficient micebetween human CD20 (7D8) IgG4 and the indicated human EGFR (2F8) IgG1and IgG4 mutant antibodies was determined by an ELISA. The presentedgraph shows average numbers (n=4). Bispecific reactivity is presented asthe concentration bispecific antibodies relative to the total IgGconcentration (percentage). Human IgG4 with a stabilized hinge (CPPC) orR409K mutation in the CH3 domain is not able to participate in Fab-armexchange. IgG1 with both a CPSC sequence in the hinge and a K409Rmutation in the CH3 domain is engaged for Fab-arm exchange. (*)Bispecific binding for the mixtures containing either IgG1-2F8,IgG4-2F8-CPPC or IgG4-2F8-R409K was below the detection limit andtherefore arbitrarily set to zero.

FIG. 5: Generation of bispecific antibodies using2-mercaptoethylamine.HCl-(2-MEA-) induced Fab-arm exchange between humanIgG1 and IgG4 antibodies. The generation of bispecific antibodies after2-MEA-induced in vitro Fab-arm exchange between the indicated human EGFR(2F8) and CD20 (7D8) antibodies was determined by an ELISA. Aconcentration series of 0-40 mM 2-MEA was tested. The presented graphshows the result of the ELISA in which a total antibody concentration of20 μg/mL was used. 2-MEA efficiently induced Fab-arm exchange, alsobetween antibodies containing a stabilized hinge (CPPC). Concerning theCH3 domains, a combination of human IgG4×human IgG1 with the triplemutation T350I-K370T-F405L, resulted in higher levels of bispecificreactivity compared to two wild type IgG4 antibodies.

FIGS. 6A and 6B: Generation of bispecific antibodies using 2-MEA-inducedFab-arm exchange between human IgG1 and IgG4 antibodies.

The generation of bispecific antibodies after 2-MEA-induced in vitroFab-arm exchange between the indicated human EGFR (2F8) and CD20 (7D8)antibodies was determined by mass spectrometry for all samples of theconcentration series of 0-40 mM 2-MEA. (FIG. 6A) Representative examplesof the mass spectrometry profiles for samples of Fab-arm exchangereactions between IgG1-2F8-ITL×IgG4-7D8-CPPC with 0 mM, 7 mM and 40 mM2-MEA are shown. (FIG. 6B) After quantification of the mass spectrometrydata, the percentage bispecific antibody was calculated and plottedagainst the concentration 2-MEA in the Fab-arm exchange reaction.IgG4-2F8×IgG4-7D8 resulted in ˜50% bispecific antibody.IgG1-2F8-ITL×IgG4-7D8-CPPC resulted in ˜95% bispecific antibody.

FIGS. 7A and 7B: Stability analysis of heterodimeric bispecificantibodies obtained by 2-MEA-induced Fab-arm exchange. The stability ofbispecific samples generated by 2-MEA induced Fab-arm exchange bycombining either IgG1-2F8-ITL×IgG4-7D8-CPPC (FIG. 7A), orIgG4-2F8×IgG4-7D8 (FIG. 7B) was tested by measuring EGFR/CD20 bispecificbinding in an ELISA after a GSH-induced Fab-arm exchange reaction in thepresence of the indicated concentrations irrelevant IgG4. Bispecificbinding is presented relative to the bispecific binding of the startingmaterial (control), which was set to 100%. (FIG. 7A) Bispecific bindingof the 2-MEA-induced bispecific product derived fromIgG1-2F8-ITL×IgG4-7D8-CPPC was preserved, indicating a stable productthat did not participate in Fab-arm exchange under GSH conditions. (FIG.7B) Bispecific EGFR/CD20 binding of the 2-MEA-induced bispecific productderived from IgG4-2F8×IgG4-7D8 was diminished, indicating that theproduct participated in Fab-arm exchange with the irrelevant IgG4 underGSH conditions.

FIGS. 8A and 8B: Plasma clearance rate of a heterodimeric bispecificantibody generated by 2-MEA-induced Fab-arm exchange. Three groups ofmice (3 mice per group) were injected with the indicated antibodies: (1)100 μg bispecific antibody, generated by in vitro 2-MEA-induced Fab-armexchange between IgG1-2F8-ITL×IgG4-7D8-CPPC; (2) 100 μg bispecificantibody+1,000 μg irrelevant IgG4; (3) 50 μg IgG1-2F8-ITL+50 μgIgG4-7D8-CPPC. (FIG. 8A) Total antibody concentrations over time,determined by ELISA. The curves of the total antibody plasmaconcentrations were the same for all antibodies. (FIG. 8B) Bispecificantibody concentration as determined by an ELISA. The bispecificity ofthe injected antibody was the same with and without the addition of anexcess irrelevant IgG4. (*) Bispecific binding for theIgG1-2F8-ITL+IgG4-7D8-CPPC mixture was below the detection limit andtherefore the corresponding symbols could not be plotted in this graph.Mean values of two ELISA experiments are shown.

FIGS. 9A-9C: Purity of bispecific antibody generated by Fab-arm exchangebetween human IgG1-2F8 and IgG4-7D8-CPPC. (FIG. 9A) Reducing SDS-PAGE(a) shows bands of the heavy and light chains for both the bispecificsample and the IgG1 control sample. Non-reducing SDS-PAGE (b). (FIG. 9B)The peak results from the HP-SEC analysis shows that >98% of thebispecific sample is homogenous, and practically no antibody aggregateswere detectable. (FIG. 9C) Mass spectrometry shows that Fab-arm exchangeresulted in approximately 100% bispecific product.

FIGS. 10A-10C: Comparison between triple mutant (ITL), double mutants(IT, IL, TL) and single mutant (L) human IgG1-2F8 in the generation ofbispecific antibodies by Fab-arm exchange with human IgG4-7D8. Thegeneration of bispecific antibodies after 2-MEA-induced in vitro Fab-armexchange between the human IgG1-2F8 triple and double mutants and wildtype IgG4-7D8 with a CPSC hinge (FIG. 10A) or mutant IgG4-7D8-CPPC witha stabilized hinge (FIG. 10B), or the single mutant IgG1-2F8-F405L andIgG4-7D8 with a wild type CPSC or stabilized CPPC hinge (FIG. 10C), wasdetermined by an ELISA. A concentration series (total antibody) of 0-20μg/mL or 0-10 μg/mL was analyzed in the ELISA for the experimentsincluding the double and single mutants, respectively. Combinations withthe double mutants IgG1-2F8-IL and -TL result in bispecific EGFR/CD20binding similar as the triple mutant IgG1-ITL. Combinations with theIgG1-2F8-IT do not result in a bispecific product. Combinations with thesingle mutant IgG1-2F8-F405L result in bispecific EGFR/CD20 binding.

FIG. 11: Generation of bispecific antibodies using 2-MEA-induced Fab-armexchange at different temperatures. The generation of bispecificantibodies by combining the indicated human EGFR (2F8) and CD20 (7D8)antibodies in 2-MEA-induced in vitro Fab-arm exchange reactions at 0°C., 20° C. and 37° C. was followed in time by an ELISA. Bispecificbinding was most efficient at 37° C., and slower at 20° C. At 0° C., nogeneration of bispecific binding was measured.

FIG. 12: Generation of bispecific antibodies by in vitro Fab-armexchange induced by different reducing agents. An ELISA was used tomeasure the generation of bispecific antibodies by combining humanIgG1-2F8-ITL and IgG4-7D8-CPPC in a reduction reaction withconcentration series of the indicated reducing agents. Bispecificbinding was measured after the reactions with DTT (maximum obtained at2.5 mM DTT) and 2-MEA (maximum obtained at 25 mM 2-MEA), but not withGSH. (*) Data for GSH concentration >10 mM were excluded due to theformation of antibody aggregates.

FIGS. 13A and 13B: 2-MEA-induced Fab-arm exchange between IgG1-2F8-ITLand IgG1-7D8-K409X mutants. The generation of bispecific antibodiesafter 2-MEA-induced in vitro Fab-arm exchange between IgG1-2F8-ITL andthe indicated IgG1-7D8-K409X mutants was determined by an ELISA. (FIG.13A) A concentration series (total antibody) of 0-20 μg/mL was analyzed.The positive control is a purified batch of bispecific antibody, derivedfrom IgG1-2F8-ITL×IgG4-7D8-CPPC. (FIG. 13B) The exchange is presented asbispecific binding at 20 μg/mL relative to the positive control (blackbar). Dark grey bars represents the bispecific binding between the IgG4control (IgG4-7D8×IgG4-2F8), the negative control(IgG1-2F8×IgG1-7D8-K409R) and between IgG1-2F8-ITL and IgG4-7D8-CPPC.Light grey bars represent results from simultaneously performedFab-arm-exchange reactions between the indicated IgG1-7D8-K409X mutantsand IgG1-2F8-ITL.

FIG. 14: Antibody deglycosylation does not affect the generation ofbispecific antibodies by 2-MEA-induced Fab-arm exchange. The generationof bispecific antibodies after 2-MEA-induced in vitro Fab-arm exchangebetween the indicated EGFR (2F8) and CD20 (7D8) antibodies wasdetermined by an ELISA. Exchange with the 7D8 antibodies was comparedwith their enzymatically deglycosylated variants. A concentration series(total antibody) of 0-20 μg/mL was analyzed in the ELISA. Fab-armexchange reactions involving deglycosylated (deglyc) antibodies showedidentical bispecific binding curves as the glycosylated variants fromwhich they were derived.

FIGS. 15A-15E: The ability to engage in Fab-arm exchange is correlatedto the CH3-CH3 interaction strength. (FIG. 15A), (FIG. 15B) and (FIG.15C) Generation of bispecific antibodies by GSH-induced Fab-arm exchangebetween IgG1-2F8 and IgG1-7D8 (FIG. 15A) or IgG4-2F8 and IgG4-7D8 (FIG.15B and FIG. 15C) constructs with the indicated mutations, presented asbispecific binding in an ELISA over time. Bispecificity is presentedrelative to the IgG4-2F8×IgG4-7D8 control after 24 h. (FIG. 15D) and(FIG. 15E) Relation between apparent K_(D) (Table 2) and bispecificantibody generation after 24 hrs (FIGS. 15A/B/C) for IgG1-based (FIG.15D) or IgG4-based (FIG. 15E) molecules.

FIG. 16: Sequence alignment of anti-EGFr antibody 2F8 in an IgG1, IgG4and (partial) IgG3 backbone. Amino acid numbering according to Kabat andaccording to the EU-index are depicted (both described in Kabat et al.,Sequences of Proteins of Immunological Interest, 5th Ed. Public HealthService, National Institutes of Health, Bethesda, Md. (1991)).

FIG. 17: Generation of bispecific antibodies by in vitroFab-arm-exchange induced by different reducing agents. An ELISA was usedto measure the generation of bispecific antibodies by combining humanIgG1-2F8-F405L and IgG1-7D8-K409R in a reduction reaction withconcentration series of the indicated reducing agents. Measured ODvalues were normalized to the signal of a bispecific control samplederived from 2-MEA-induced Fab-arm-exchange betweenIgG1-2F8-ITL×IgG4-7D8-CPPC, which was set to 100%. Maximal bispecificbinding was measured after the reactions with DTT in the concentrationrange 0.5-50 mM, 2-MEA in the concentration range 25-50 mM andtris(2-carboxyethyl)phosphine (TCEP) in the concentration range 0.5-5.0mM, but not with GSH. (*) Data for GSH concentration ≧25 mM wereexcluded due to the formation of antibody aggregates.

FIGS. 18A and 18B: Generation of bispecific antibodies using2-MEA-induced Fab-arm-exchange between human IgG1-2F8-F405L andIgG1-7D8-K409R.

(FIG. 18A) The generation of bispecific antibodies after 2-MEA-inducedin vitro Fab-arm-exchange was determined by an ELISA. The presentedgraph shows the result of the ELISA in which a total antibodyconcentration of 20 μg/mL was used. 2-MEA efficiently inducedFab-arm-exchange. (FIG. 18B) The generation of bispecific antibodiesafter 2-MEA-induced in vitro Fab-arm-exchange was determined by massspectrometry for all samples of the concentration series of 0-40 mM2-MEA. After quantification of the mass spectrometry data, thepercentage bispecific antibody was calculated and plotted against theconcentration of 2-MEA in the Fab-arm-exchange reaction.IgG1-2F8-F405L×IgG1-7D8-K409R resulted in ˜100% bispecific antibody,confirming the ELISA data.

FIG. 19: Purity of bispecific antibody generated by Fab-arm-exchangebetween human IgG1-2F8-F405L×IgG1-7D8-K409R. Mass spectrometry showsthat Fab-arm-exchange resulted in approximately 100% bispecific product.

FIGS. 20A and 20B: Plasma clearance of a bispecific antibody generatedby 2-MEA-induced Fab-arm-exchange. Two groups of mice (3 mice per group)were injected with the indicated antibodies: (1) 100 μg bispecificantibody, generated by in vitro 2-MEA-induced Fab-arm-exchange betweenIgG1-2F8-F405L×IgG1-7D8-K409R; (2) 100 μg bispecific antibody+1,000 μgirrelevant IgG4. (FIG. 20A) Total antibody concentrations over time,determined by ELISA. The curves of the total antibody plasmaconcentrations were the same for all antibodies. (FIG. 20B) Bispecificantibody concentration as determined by an ELISA. The bispecificity ofthe injected antibody was the same with and without the addition of anexcess irrelevant IgG4.

FIGS. 21A and 21B: CDC-mediated cell kill of CD20-expressing cells by abispecific antibody generated by 2-MEA-induced Fab-arm-exchange betweenIgG1-2F8-F405L×IgG1-7D8-K409R. Concentration series of the indicatedantibodies were used to test their capacity to mediate CDC on Daudi(FIG. 21A) and Raji (FIG. 21B) cells. Both cell lines express CD20 butnot EGFR. Introduction of the K409R in IgG1-7D8 did not influence itscapacity to induce CDC. The bispecific antibody derived from2-MEA-induced Fab-arm-exchange between IgG1-2F8-F405L×IgG1-7D8-K409R wasstill capable to induce CDC.

FIG. 22: ADCC-mediated cell kill of EGFR-expressing cells by abispecific antibody generated by 2-MEA-induced Fab-arm-exchange betweenIgG1-2F8-F405L×IgG1-7D8-K409R. Concentration series of the indicatedantibodies were used to test their capacity to mediate ADCC on A431cells. IgG1-7D8 can not bind the CD20-negative A431 cells andconsequently did not induce ADCC. ADCC was induced by the EGFR antibodyIgG1-2F8, also after introduction of the F405L mutations in the CH3domain. The ADCC effector function of IgG1-2F8-F405L was retained in thebispecific format obtained by Fab-arm-exchange betweenIgG1-2F8-F405L×IgG1-7D8-K409R.

FIGS. 23A and 23B: 2-MEA-induced Fab-arm-exchange between IgG1-2F8-F405Xmutants and IgG1-7D8-K409R. The generation of bispecific antibodiesafter 2-MEA-induced in vitro Fab-arm-exchange between the indicatedIgG1-2F8-F405X mutants and IgG1-7D8-K409R was determined by an ELISA.(FIG. 23A) A concentration series (total antibody) of 0-20 μg/mL wasanalyzed in the ELISA. The positive control is a purified batch ofbispecific antibody, derived from IgG1-2F8-F405L×IgG1-7D8-K409R. (FIG.23B) The exchange is presented as bispecific binding at 20 μg/mLantibody concentration relative to the positive control (black bar).Dark grey bars represents the bispecific binding between the IgG4control (IgG4-7D8×IgG4-2F8) and the negative control(IgG1-2F8×IgG1-7D8-K409R). Light grey bars represent results fromsimultaneously performed Fab-arm-exchange reactions between theindicated IgG1-2F8-F405X mutants and IgG1-7D8-K409R or controls.

FIGS. 24A and 24B: 2-MEA-induced Fab-arm-exchange between IgG1-2F8-Y407Xmutants and IgG1-7D8-K409R. The generation of bispecific antibodiesafter 2-MEA-induced in vitro Fab-arm-exchange between the indicatedIgG1-2F8-Y407X mutants and IgG1-7D8-K409R was determined by an ELISA.(FIG. 24A) A concentration series (total antibody) of 0-20 μg/mL wasanalyzed in the ELISA. The positive control is a purified batch ofbispecific antibody, derived from IgG1-2F8-F405L×IgG1-7D8-K409R. (FIG.24B) The exchange is presented as bispecific binding at 20 μg/mLantibody concentration relative to the positive control (black bar).Dark grey bars represents the bispecific binding between the IgG4control (IgG4-7D8×IgG4-2F8) and the negative control(IgG1-2F8×IgG1-7D8-K409R). Light grey bars represent results fromsimultaneously performed Fab-arm-exchange reactions between theindicated IgG1-2F8-Y407X mutants and IgG1-7D8-K409R or controls.

FIGS. 25A and 25B: Analysis of bispecific antibody generated by2-MEA-induced Fab-arm exchange by SDS-PAGE under non-reducing (FIG.25(A)) and reducing (FIG. 25(B)) conditions.

FIGS. 26A-26D: HP-SEC profiles of the homodimer starting materialIgG1-2F8-F405L (FIG. 26(B)), the homodimer starting materialIgG1-7D8-K409R (FIG. 26(A)), the mixture (1:1) of both homodimers (FIG.26(C)), and the bispecific product generated by 2-MEA-induced Fab-armexchange between IgG1-2F8-F405L×IgG1-7D8-K409R (FIG. 26(D)).

FIGS. 27A-27D: Mass spectrometry (ESI-MS) of the homodimer startingmaterial IgG1-2F8-F405L (FIG. 27(B)), the homodimer starting materialIgG1-7D8-K409R (FIG. 27(A)), the mixture (1:1) of both homodimers (FIG.27(C)), and the bispecific product generated by 2-MEA-induced Fab-armexchange between IgG1-2F8-F405L×IgG1-7D8-K409R (FIG. 27(D)).

FIGS. 28A-28D: Capillary isoelectrofocussing (cIEF) profiles of thehomodimer starting material IgG1-2F8-F405L (FIG. 28(A)), the homodimerstarting material IgG1-7D8-K409R (FIG. 28(B)), the mixture (1:1) of bothhomodimers (FIG. 28(C)), and the bispecific product generated by2-MEA-induced Fab-arm exchange between IgG1-2F8-F405L×IgG1-7D8-K409R(FIG. 28(D)).

FIGS. 29A-29D: HPLC-CIEX profiles of the homodimer starting materialIgG1-2F8-F405L (FIG. 29(A)), the homodimer starting materialIgG1-7D8-K409R (FIG. 29(B)), the mixture (1:1) of both homodimers (FIG.29(C)), and the bispecific product generated by 2-MEA-induced Fab-armexchange between IgG1-2F8-F405L×IgG1-7D8-K409R (FIG. 29(D)).

FIG. 30: Electrospray ionization mass spec analysis of IgG obtained byco-transfection of the expression vectors encoding the heavy and lightchain of IgG1-7D8-K409R or IgG1-2F8-F405. Heterodimer peaks areindicated with an *. Homodimer peaks are indicated with an †.

FIGS. 31A-31H: Exchange reaction of the homodimers IgG1-2F8-F405L andIgG1-7D8-K409R as monitored by High Pressure Liquid ChromatographyCation Exchange (HPLC-CIEX) after injection at different intervals.

FIGS. 32A and 32B: Residual homodimers of the exchange reaction as shownin FIG. 32 as detected with the CIEX method (indicated by arrows).

FIGS. 33A-33F: Generation of bispecific antibodies at various IgGconcentrations, 2-MEA concentrations, incubation temperatures and timesas determined by an ELISA.

FIGS. 34A-34D: Generation of bispecific antibodies at various IgGconcentrations, 2-MEA concentrations, incubation temperatures and timesas determined by an ELISA and compared to control which was arbitrarilyset to 100%.

FIGS. 35A-35D: Generation of bispecific antibodies at various IgGconcentrations, 2-MEA concentrations, incubation temperatures and timesas analysed by HPLC-CIEX.

FIGS. 36A and 36B: Generation of bispecific antibodies after2-MEA-induced in vitro Fab-arm exchange between the indicatedIgG1-2F8-L368X mutants and IgG1-7D8-K409R was determined by an ELISAusing a concentration series (total antibody) of 0-20 μg/mL (FIG. 36A).The positive control is a purified batch of bispecific antibody, derivedfrom IgG1-2F8-F405L×IgG1-7D8-K409R. FIG. 36B shows the bispecificbinding at 20 μg/mL relative to the positive control (black bar). Darkgrey bars represents the bispecific binding between the IgG4 control(IgG4-7D8×IgG4-2F8) and the negative control (IgG1-2F8×IgG1-7D8-K409R).Light grey bars represent results from simultaneously performedFab-arm-exchange reactions between the indicated IgG1-2F8-L368X mutantsand IgG1-7D8-K409R.

FIGS. 37A and 37B: Generation of bispecific antibodies after2-MEA-induced in vitro Fab-arm exchange between the indicatedIgG1-2F8-K370X mutants and IgG1-7D8-K409R was determined by an ELISAusing a concentration series (total antibody) of 0-20 μg/mL (FIG.37(A)). The positive control is a purified batch of bispecific antibody,derived from IgG1-2F8-F405L×IgG1-7D8-K409R. FIG. 37(B) shows thebispecific binding at 20 μg/mL relative to the positive control (blackbar). Dark grey bars represents the bispecific binding between the IgG4control (IgG4-7D8×IgG4-2F8) and the negative control(IgG1-2F8×IgG1-7D8-K409R). Light grey bars represent results fromsimultaneously performed Fab-arm-exchange reactions between theindicated IgG1-2F8-D370X mutants and IgG1-7D8-K409R.

FIGS. 38A and 38B: Generation of bispecific antibodies after2-MEA-induced in vitro Fab-arm exchange between the indicatedIgG1-2F8-D399X mutants and IgG1-7D8-K409R was determined by an ELISAusing a concentration series (total antibody) of 0-20 μg/mL (FIG.38(A)). FIG. 38(B) shows the bispecific binding at 20 μg/mL antibodyconcentration relative to the positive control (black bar). Dark greybars represents the bispecific binding between the IgG4 control(IgG4-7D8×IgG4-2F8) and the negative control (IgG1-2F8×IgG1-7D8-K409R).Light grey bars represent results from simultaneously performedFab-arm-exchange reactions between the indicated IgG1-2F8-D399X mutantsand IgG1-7D8-K409R.

FIG. 39: 2-MEA-induced Fab-arm exchange between four different IgG1mutant combinations at 15° C. after 0, 30, 60, 105 and 200 minincubations as determined by sandwich ELISA.

FIG. 40: 2-MEA-induced Fab-arm exchange between different IgG1 mutantcombinations after antibody incubation at 15° C. for 90 min asdetermined by sandwich ELISA.

FIG. 41: Phosphorylation of c-Met by c-Met specific antibodies. A549cells are incubated for 15 min with HGF or a panel of differentantibodies. Proteins are separated by SDS-page gel electrophoresis andtransferred to membranes by western blotting. Phosphorylated c-Met,total c-Met and β-actin are detected by antibodies againstphosphorylated c-Met, total c-Met or β-actin.

FIG. 42: Proliferation assay with NCI-H441 cells. NCI-H441 cells wereincubated for seven days with monovalent bispecific IgG1 069/b12,control antibodies (IgG1-069, UniBody-069, IgG1-b12) left untreated.Cell mass was determined and plotted as percentage of non-treatedsamples (set as 100%)

FIGS. 43A and 43B: CDC-mediated cell kill of CD20 expressing cells bybispecific antibodies generated by 2-MEA-induced Fab-arm-exchangebetween IgG1-7D8-F405L or IgG1-2F8-F405L and IgG1-7D8-K409R.Concentration series of the indicated antibodies were used to test theircapacity to mediate CDC on Daudi (FIG. 43A) and Raji (FIG. 43B) cells.Both cell lines express CD20 but not EGFR. The bispecific antibodygenerated by 2-MEA-induced Fab-arm-exchange betweenIgG1-7D8-F405L×IgG1-7D8-K409R was as effective as IgG1-7D8 in inductionof CDC mediated cell kill. The bispecific antibody derived from2-MEA-induced Fab-arm-exchange between IgG2-2F8-F405L×IgG1-7D8-K409Rresults in a monovalent CD20 binding bispecific antibody, which slightlyaffected the induction of CDC mediated cell kill with slightly.

FIG. 44: Killing of A431 cells induced by anti-kappa-ETA′ pre-incubatedHER2×HER2 bispecific antibodies. The viability of A431 cells after 3days incubation with HER2 antibodies, pre-incubated withanti-kappa-ETA′. Cell viability was quantified using Alamarblue. Datashown are fluorescence intensities (FI) of one experiment with A431cells treated with anti-kappa-ETA′-conjugated HER2 antibodies andHER2×HER2 bispecific antibodies. Staurosporin was used as positivecontrol, whereas an isotype control antibody was used as negativecontrol.

FIG. 45: HER2×HER2 bispecific molecules induced downmodulation of HER2receptor. Relative percentage of HER2 expression levels in AU565 celllysates after 3 days incubation with 10 μg/mL mAb. The amount of HER2was quantified using a HER2-specific capture ELISA and depicted aspercentage inhibition compared to untreated cells. Data shown is themean of two experiments plus standard deviation.

FIGS. 46A and 46B: Colocalization analysis of HER2×HER2 bispecificantibodies (FITC) with lysosomal marker LAMP1 (Cy5). FITC pixelintensity overlapping with Cy5 for various monospecific HER2 antibodiesand HER2×HER2 bispecific antibodies (FIG. 46A) FITC pixel intensity inLAMP1/Cy5 positive pixels of three different images is plotted for eachantibody tested. Monospecifics show lower FITC pixel intensities in theLAMP1/Cy5 positive pixels compared to bispecifics. FIG. 46(B) representsthe mean value of FITC pixel intensity per LAMP1/Cy5 positive pixelcalculated from the three different images. Together these resultsindicate that after internalization higher levels of bispecificantibodies, compared to monospecifics antibodies, localize to Lamp1/Cy5positive vesicles.

FIG. 47: Inhibition of proliferation by HER-2 mono and bispecificantibodies. AU565 cells were seeded in the presence of 10 μg/mL HER2antibody or HER2×HER2 bispecific antibody in serum-free cell culturemedium. After three days, the amount of viable cells was quantified withAlamarblue and cell viability was presented as a percentage relative tountreated cells. An isotype control antibody was used as negativecontrol. Data shown are percentage viable AU565 cells compared tountreated cells measured in five-fold±the standard deviation. *indicates only one data point was depicted.

FIGS. 48A and 48B: Binding of mono and bispecific IgG1 and hinge-deletedIgG1 antibodies to human and mouse FcRn at different pH. Plates withhuman and mouse FcRn were incubated with different mono- and bispecificIgG1 antibodies or hinge-deleted IgG1 molecules. Binding to FcRn wasanalyzed by ELISA at 405 nm. (FIG. 48A) Binding of mono and bispecificIgG1 antibodies and hinge-deleted IgG1 (Uni-G1) molecules to human FcRnat pH 7.4 and 6.0. Binding to human FcRn is very low at neutral pH. AtpH 6.0 (bispecific) antibodies bind efficiently to human FcRn, unlessthey contain the H435A mutation. Hinge-deleted IgG1 (Uni-G1) moleculesbind human FcRn with low efficiency. (FIG. 48B) Binding of mono andbispecific IgG1 antibodies and hinge-deleted IgG1 (Uni-G1) molecules tomouse FcRn at pH 7.4 and 6.0. Binding to mouse FcRn is very low atneutral pH. At pH 6.0 (bispecific) antibodies bind very efficiently tomouse FcRn, unless they contain the H435A mutation in both Fab-arms. Thebispecific molecule harboring the H435A mutation in only one Fab-arm isstill able to bind mouse FcRn. Hinge-deleted IgG1 (Uni-G1) moleculesbind mouse FcRn with intermediate efficiency and the hinge-deleted IgG1(Uni-G1) bispecific molecule harboring the H435A mutation in only oneFab-arm is slightly less efficient.

FIG. 49: T cell mediated cytotoxicity of AU565 cells by Her2×CD3bispecific antibodies as well as by N297Q mutants of Her2×CD3 bispecificantibodies.

DETAILED DESCRIPTION OF THE INVENTION Definitions

The term “immunoglobulin” refers to a class of structurally relatedglycoproteins consisting of two pairs of polypeptide chains, one pair oflight (L) low molecular weight chains and one pair of heavy (H) chains,all four inter-connected by disulfide bonds. The structure ofimmunoglobulins has been well characterized. See for instanceFundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y.(1989)). Briefly, each heavy chain typically is comprised of a heavychain variable region (abbreviated herein as VH) and a heavy chainconstant region. The heavy chain constant region typically is comprisedof three domains, CH1, CH2, and CH3. The heavy chains areinter-connected via disulfide bonds in the so-called “hinge region”.Each light chain typically is comprised of a light chain variable region(abbreviated herein as VL) and a light chain constant region. The lightchain constant region typically is comprised of one domain, CL.Typically, the numbering of amino acid residues in the constant regionis performed according to the EU-index as described in Kabat et al.,Sequences of Proteins of Immunological Interest, 5th Ed. Public HealthService, National Institutes of Health, Bethesda, Md. (1991). FIG. 16gives an overview of the EU and Kabat numbering for different isotypeforms of antibody 2F8 (WO 02/100348). The VH and VL regions may befurther subdivided into regions of hypervariability (or hypervariableregions which may be hypervariable in sequence and/or form ofstructurally defined loops), also termed complementarity determiningregions (CDRs), interspersed with regions that are more conserved,termed framework regions (FRs). Each VH and VL is typically composed ofthree CDRs and four FRs, arranged from amino-terminus tocarboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4 (see also Chothia and Lesk J. Mol. Biol. 196, 901 917 (1987)).

When used herein, the term “Fab-arm” refers to one heavy chain-lightchain pair.

When used herein, the term “Fc region” refers to an antibody regioncomprising at least the hinge region, a CH2 domain and a CH3 domain.

The term “antibody” (Ab) in the context of the present invention refersto an immunoglobulin molecule, a fragment of an immunoglobulin molecule,or a derivative of either thereof, which has the ability to specificallybind to an antigen under typical physiological conditions with a halflife of significant periods of time, such as at least about 30 min, atleast about 45 min, at least about one hour, at least about two hours,at least about four hours, at least about 8 hours, at least about 12hours (h), about 24 hours or more, about 48 hours or more, about 3, 4,5, 6, 7 or more days, etc., or any other relevant functionally-definedperiod (such as a time sufficient to induce, promote, enhance, and/ormodulate a physiological response associated with antibody binding tothe antigen and/or time sufficient for the antibody to recruit aneffector activity). The variable regions of the heavy and light chainsof the immunoglobulin molecule contain a binding domain that interactswith an antigen. The constant regions of the antibodies (Abs) maymediate the binding of the immunoglobulin to host tissues or factors,including various cells of the immune system (such as effector cells)and components of the complement system such as C1q, the first componentin the classical pathway of complement activation. An antibody may alsobe a bispecific antibody, diabody, or similar molecule. The term“bispecific antibody” refers to antibody having specificities for atleast two different epitopes, typically non-overlapping epitopes. Asindicated above, the term antibody herein, unless otherwise stated orclearly contradicted by the context, includes fragments of an antibodythat retain the ability to specifically bind to the antigen. Suchfragments may be provided by any known technique, such as enzymaticcleavage, peptide synthesis and recombinant expression techniques. Ithas been shown that the antigen-binding function of an antibody may beperformed by fragments of a full-length antibody, e.g. a F(ab′)2fragment. It also should be understood that the term antibody, unlessspecified otherwise, also includes polyclonal antibodies, monoclonalantibodies (mAbs), antibody-like polypeptides, such as chimericantibodies and humanized antibodies. An antibody as generated canpossess any isotype.

The term “full-length antibody” when used herein, refers to an antibodywhich contains all heavy and light chain constant and variable domainsthat are normally found in an antibody of that isotype.

As used herein, “isotype” refers to the immunoglobulin class (forinstance IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM) that is encodedby heavy chain constant region genes.

The term “human antibody”, as used herein, is intended to includeantibodies having variable and constant regions derived from humangermline immunoglobulin sequences. The human antibodies of the inventionmay include amino acid residues not encoded by human germlineimmunoglobulin sequences (e.g., mutations introduced by random orsite-specific mutagenesis in vitro or by somatic mutation in vivo).However, the term “human antibody”, as used herein, is not intended toinclude antibodies in which CDR sequences derived from the germline ofanother mammalian species, such as a mouse, have been grafted onto humanframework sequences.

When used herein, the term “heavy chain antibody” or “heavy-chainantibody” refers to an antibody which consists only of two heavy chainsand lacks the two light chains usually found in antibodies. Heavy chainantibodies, which naturally occur in e.g. camelids, can bind antigensdespite having only VH domains.

The term “epitope” means a protein determinant capable of specificbinding to an antibody. Epitopes usually consist of surface groupings ofmolecules such as amino acids or sugar side chains and usually havespecific three dimensional structural characteristics, as well asspecific charge characteristics. Conformational and nonconformationalepitopes are distinguished in that the binding to the former but not thelatter is lost in the presence of denaturing solvents. The epitope maycomprise amino acid residues directly involved in the binding (alsocalled immunodominant component of the epitope) and other amino acidresidues, which are not directly involved in the binding, such as aminoacid residues which are effectively blocked by the specifically antigenbinding peptide (in other words, the amino acid residue is within thefootprint of the specifically antigen binding peptide).

As used herein, the term “binding” in the context of the binding of anantibody to a predetermined antigen typically is a binding with anaffinity corresponding to a K_(D) of about 10⁻⁶ M or less, e.g. 10⁻⁷ Mor less, such as about 10⁻⁸ M or less, such as about 10⁻⁹ M or less,about 10⁻¹⁰ M or less, or about 10⁻¹¹ M or even less when determined byfor instance surface plasmon resonance (SPR) technology in a BIAcore3000 instrument using the antigen as the ligand and the antibody as theanalyte, and binds to the predetermined antigen with an affinitycorresponding to a K_(D) that is at least ten-fold lower, such as atleast 100 fold lower, for instance at least 1,000 fold lower, such as atleast 10,000 fold lower, for instance at least 100,000 fold lower thanits affinity for binding to a non-specific antigen (e.g., BSA, casein)other than the predetermined antigen or a closely-related antigen. Theamount with which the affinity is lower is dependent on the K_(D) of theantibody, so that when the K_(D) of the antibody is very low (that is,the antibody is highly specific), then the amount with which theaffinity for the antigen is lower than the affinity for a non-specificantigen may be at least 10,000 fold. The term “K_(D)” (M), as usedherein, refers to the dissociation equilibrium constant of a particularantibody-antigen interaction.

When used herein the term “heterodimeric interaction between the firstand second CH3 regions” refers to the interaction between the first CH3region and the second CH3 region in a first-CH3/second-CH3 heterodimericprotein.

When used herein the term “homodimeric interactions of the first andsecond CH3 regions” refers to the interaction between a first CH3 regionand another first CH3 region in a first-CH3/first-CH3 homodimericprotein and the interaction between a second CH3 region and anothersecond CH3 region in a second-CH3/second-CH3 homodimeric protein.

An “isolated antibody,” as used herein, denotes that the material hasbeen removed from its original environment (e.g., the naturalenvironment if it is naturally occurring or the host cell if it isrecombinantly expressed). It is also advantageous that the antibodies bein purified form. The term “purified” does not require absolute purity;rather, it is intended as a relative definition, indicating an increaseof the antibody concentration relative to the concentration ofcontaminants in a composition as compared to the starting material.

The term “host cell”, as used herein, is intended to refer to a cellinto which an expression vector has been introduced, e.g. an expressionvector encoding an antibody of the invention. Recombinant host cellsinclude, for example, transfectomas, such as CHO cells, HEK293 cells,NS/0 cells, and lymphocytic cells.

When used herein, the term “co-expression” of two or more nucleic acidconstructs, refers to expression of the two constructs in a single hostcell.

The term “tumor cell protein” refers to a protein located on the cellsurface of a tumor cell.

As used herein, the term “effector cell” refers to an immune cell whichis involved in the effector phase of an immune response, as opposed tothe cognitive and activation phases of an immune response. Exemplaryimmune cells include a cell of a myeloid or lymphoid origin, forinstance lymphocytes (such as B cells and T cells including cytolytic Tcells (CTLs)), killer cells, natural killer cells, macrophages,monocytes, eosinophils, polymorphonuclear cells, such as neutrophils,granulocytes, mast cells, and basophils. Some effector cells expressspecific Fc receptors and carry out specific immune functions. In someembodiments, an effector cell is capable of inducing antibody-dependentcellular cytotoxicity (ADCC), such as a natural killer cell, capable ofinducing ADCC. In some embodiments, an effector cell may phagocytose atarget antigen or target cell.

The term “reducing conditions” or “reducing environment” refers to acondition or an environment in which a substrate, here a cysteineresidue in the hinge region of an antibody, is more likely to becomereduced than oxidized.

The term “disulfide bond isomerization” refers to an exchange ofdisulfide bonds between different cysteines, i.e., the shuffling ofdisulfide bonds.

Further Aspects and Embodiments of the Invention

As described above, in a first aspect, the invention relates to an invitro method for generating a heterodimeric protein, said methodcomprising the following steps:

a) providing a first homodimeric protein comprising an Fc region of animmunoglobulin, said Fc region comprising a first CH3 region,

b) providing a second homodimeric protein comprising an Fc region of animmunoglobulin, said Fc region comprising a second CH3 region,

wherein the sequences of said first and second CH3 regions are differentand are such that the heterodimeric interaction between said first andsecond CH3 regions is stronger than each of the homodimeric interactionsof said first and second CH3 regions,

c) incubating said first protein together with said second protein underreducing conditions sufficient to allow the cysteines in the hingeregion to undergo disulfide-bond isomerization, and

d) obtaining said heterodimeric protein.

The bispecific format may be used in many ways to generate desiredcombinations of bispecific antibodies. In addition to being able ofcombining antibodies targeting different antigens in a very selectiveway it can be used to change a desired property, e.g. to increase CDC,by combining two different antibodies targeting the same antigen.Furthermore, it can be used to remove partial agonistic activity of anantagonistic antibody or convert an agonistic antibody into anantagonistic antibody by making a bispecific antibody thereof with anirrelevant (inactive) antibody.

In one embodiment, the homodimeric proteins are selected from the groupconsisting of (i) an Fc region, (ii) an antibody, (iii) a fusion proteincomprising an Fc region, such as an Fc region fused to a receptor,cytokine or hormone, and (iv) a Fc region conjugated to a prodrug,peptide, drug or a toxin.

In some embodiments, said first and/or second homodimeric proteincomprise, in addition to the Fc region, one or more or all of the otherregions of an antibody, i.e. a CH1 region, a VH region, a CL regionand/or a VL region. Thus, in one embodiment, said first homodimericprotein is a full-length antibody. In another embodiment, said secondhomodimeric protein is a full-length antibody.

In an important embodiment, said first and second homodimeric proteinsare both antibodies, preferably full-length antibodies, and binddifferent epitopes. In such an embodiment, the heterodimeric proteinthat is generated is a bispecific antibody. Said epitopes may be locatedon different antigens or on the same antigen.

In other embodiments, however, only one of the homodimeric proteins is afull-length antibody and the other homodimeric protein is not afull-length antibody, e.g. an Fc region without a variable region,expressed in conjunction to another protein or peptide sequence like areceptor, cytokine or hormone, or conjugated to a prodrug, peptide, adrug or a toxin. In a further embodiment, neither of the homodimericproteins is a full-length antibody. For example, both homodimericproteins may be Fc regions that are fused to another protein or peptidesequence like a receptor, cytokine or hormone, or conjugated to aprodrug, peptide, a drug or a toxin.

In one embodiment, the Fc region of the first homodimeric protein is ofan isotype selected from the group consisting of IgG1, IgG2, IgG3 andIgG4 and the Fc region of the second homodimeric protein is of anisotype selected from the group consisting of IgG1, IgG2, IgG3 and IgG4.In a preferred embodiment, the Fc regions of both said first and saidsecond homodimeric protein are of the IgG1 isotype. In another preferredembodiment, one of the Fc regions of said homodimeric proteins is of theIgG1 isotype and the other of the IgG4 isotype. In the latterembodiment, the resulting heterodimeric comprises an Fc region of anIgG1 and an Fc region of IgG4 and may thus have interesting intermediateproperties with respect to activation of effector functions. A similarproduct can be obtained if said first and/or said second homodimericprotein comprises a mutation removing the acceptor site for Asn-linkedglycosylation or is otherwise manipulated to change the glycosylationproperties.

In a further embodiment, one or both of the homodimeric proteins isglyco-engineered to reduce fucose and thus enhance ADCC, e.g. byaddition of compounds to the culture media during antibody production asdescribed in US2009317869 or as described in van Berkel et al. (2010)Biotechnol. Bioeng. 105:350 or by using FUT8 knockout cells, e.g. asdescribed in Yamane-Ohnuki et al (2004) Biotechnol. Bioeng 87:614. ADCCmay alternatively be optimized using the method described by Umaña etal. (1999) Nature Biotech 17:176.

In a further embodiment, one or both of the homodimeric proteins hasbeen engineered to enhance complement activation, e.g. as described inNatsume et al. (2009) Cancer Sci. 100:2411.

In a further embodiment, one or both of the homodimeric proteins hasbeen engineered to reduce or increase the binding to the neonatal Fcreceptor (FcRn) in order to manipulate the serum half-life of theheterodimeric protein.

In a further embodiment, one of the homodimeric starting proteins hasbeen engineered to not bind Protein A, thus allowing to separate theheterodimeric protein from said homodimeric starting protein by passingthe product over a protein A column. This may in particular be usefulfor embodiments wherein an excess of one homodimeric protein is usedrelative to the other homodimeric protein as starting material. In suchembodiments, it may be useful to engineer the homodimeric protein thatis in excess so that is looses its ability to bind protein A. Followingthe heterodimerization reaction, the heterodimeric protein may then beseparated from a surplus of unexchanged homodimeric protein by passageover a protein A column.

In a further embodiment, one of the homodimeric proteins is an Fc regionor a full-length antibody recognizing a non-relevant epitope or afull-length antibody containing germline-derived sequences that have notundergone somatic hypermutation and do not bind self-antigens. In suchan embodiment the heterodimeric protein functions as a monovalentantibody. In another embodiment, both homodimeric proteins comprises thesame heavy chain, but only one of the homodimeric proteins contains alight chain which forms a functional antigen-binding site with saidheavy chain, whereas the other homodimeric protein contains anon-functional light chain, which does not bind any antigen incombination with said heavy chain. In such an embodiment, theheterodimeric protein functions as a monovalent antibody. Such anon-functional light chain can e.g. be a germline-derived sequence thathas not undergone somatic hypermutation and does not bind self-antigens.

Antibodies to be used as homodimeric starting material of the presentinvention may e.g. be produced by the hybridoma method first describedby Kohler et al., Nature 256, 495 (1975), or may be produced byrecombinant DNA methods. Monoclonal antibodies may also be isolated fromphage antibody libraries using the techniques described in, for example,Clackson et al., Nature 352, 624 628 (1991) and Marks et al., J. Mol.Biol. 222, 581 597 (1991). Monoclonal antibodies may be obtained fromany suitable source. Thus, for example, monoclonal antibodies may beobtained from hybridomas prepared from murine splenic B cells obtainedfrom mice immunized with an antigen of interest, for instance in form ofcells expressing the antigen on the surface, or a nucleic acid encodingan antigen of interest. Monoclonal antibodies may also be obtained fromhybridomas derived from antibody-expressing cells of immunized humans ornon-human mammals such as rats, dogs, primates, etc.

Antibodies to be used as homodimeric starting material of the presentinvention may e.g. chimeric or humanized antibodies. In anotherembodiment, one or both of the homodimeric starting proteins, except forany specified mutations, is a human antibody. Human monoclonalantibodies may be generated using transgenic or transchromosomal mice,e.g. HuMAb mice, carrying parts of the human immune system rather thanthe mouse system. The HuMAb mouse contains a human immunoglobulin geneminiloci that encodes unrearranged human heavy (μ and γ) and κ lightchain immunoglobulin sequences, together with targeted mutations thatinactivate the endogenous μ and κ chain loci (Lonberg, N. et al., Nature368, 856 859 (1994)). Accordingly, the mice exhibit reduced expressionof mouse IgM or κ and in response to immunization, the introduced humanheavy and light chain transgenes, undergo class switching and somaticmutation to generate high affinity human IgG,κ monoclonal antibodies(Lonberg, N. et al. (1994), supra; reviewed in Lonberg, N. Handbook ofExperimental Pharmacology 113, 49 101 (1994), Lonberg, N. and Huszar,D., Intern. Rev. Immunol. Vol. 13 65 93 (1995) and Harding, F. andLonberg, N. Ann. N.Y. Acad. Sci 764 536 546 (1995)). The preparation ofHuMAb mice is described in detail in Taylor, L. et al., Nucleic AcidsResearch 20, 6287 6295 (1992), Chen, J. et al., International Immunology5, 647 656 (1993), Tuaillon et al., J. Immunol. 152, 2912 2920 (1994),Taylor, L. et al., International Immunology 6, 579 591 (1994), Fishwild,D. et al., Nature Biotechnology 14, 845 851 (1996). See also U.S. Pat.No. 5,545,806, U.S. Pat. No. 5,569,825, U.S. Pat. No. 5,625,126, U.S.Pat. No. 5,633,425, U.S. Pat. No. 5,789,650, U.S. Pat. No. 5,877,397,U.S. Pat. No. 5,661,016, U.S. Pat. No. 5,814,318, U.S. Pat. No.5,874,299, U.S. Pat. No. 5,770,429, U.S. Pat. No. 5,545,807, WO98/24884, WO 94/25585, WO 93/1227, WO 92/22645, WO 92/03918 and WO01/09187. Splenocytes from these transgenic mice may be used to generatehybridomas that secrete human monoclonal antibodies according to wellknown techniques.

Further, human antibodies of the present invention or antibodies of thepresent invention from other species may be identified throughdisplay-type technologies, including, without limitation, phage display,retroviral display, ribosomal display, mammalian display, and othertechniques, using techniques well known in the art and the resultingmolecules may be subjected to additional maturation, such as affinitymaturation, as such techniques are well known in the art.

In a further embodiment of the invention, the antibody or a partthereof, e.g. one or more CDRs, is of a species in the family Camelidae,see WO2010001251, or a species of cartilaginous fish, such as the nurseshark or heavy-chain or domain antibodies.

In one embodiment of the method of the invention, said first and secondhomodimeric proteins provided in step a) and b) are purified.

In one embodiment, the first and/or second homodimeric protein isconjugated to a drug, a prodrug or a toxin or contains an acceptor groupfor the same. Such acceptor group may e.g. be an unnatural amino acid.

As described above, the sequences of the first and second CH3 regions ofthe homodimeric starting proteins are different and are such that theheterodimeric interaction between said first and second CH3 regions isstronger than each of the homodimeric interactions of said first andsecond CH3 regions.

In one embodiment, the increased strength of the heterodimericinteraction as compared to each of the homodimeric interactions is dueto CH3 modifications other than the introduction of covalent bonds,cysteine residues or charged residues.

In some embodiments, the product of the invention is highly stable anddoes not undergo Fab-arm exchange under mildly reducing conditions invitro or, importantly, in vivo upon administration to a human being.Thus, in one embodiment, the heterodimeric interaction between saidfirst and second proteins in the resulting heterodimeric protein is suchthat no Fab-arm exchange can occur at 0.5 mM GSH under the conditionsdescribed in Example 13.

In another embodiment, the heterodimeric interaction between said firstand second proteins in the resulting heterodimeric protein is such thatno Fab-arm exchange occurs in vivo in mice under the conditionsdescribed in Example 14.

In another embodiment, the heterodimeric interaction between said firstand second proteins in the resulting heterodimeric protein is more thantwo times stronger, such as more than three times stronger, e.g. morethan five times stronger than the strongest of the two homodimericinteractions, e.g. when determined as described in Example 30.

In a further embodiment, the sequences of said first and second CH3regions are such that the dissociation constants of the heterodimericinteraction between said first and second proteins in the resultingheterodimeric protein is below 0.05 micromolar when assayed as describedin Example 30.

In a further embodiment, the sequences of said first and second CH3regions are such that the dissociation constants of both homodimericinteractions are above 0.01 micromolar, such as above 0.05 micromolarpreferably between 0.01 and 10 micromolar, such as between 0.05 and 10micromolar, more preferably between 0.01 and 5, such as between 0.05 and5 micromolar, even more preferably between 0.01 and 1 micromolar, suchas between 0.05 and 1 micromolar, between 0.01 and 0.5 or between 0.01and 0.1 when assayed as described in Example 21. Embodiments wherein thehomodimeric starting proteins are relatively stable can have theadvantage that it is easier to produce a large quantity of startingprotein and e.g. avoid aggregation or misfolding.

In some embodiments, a stable heterodimeric protein can be obtained athigh yield using the method of the invention on the basis of twohomodimeric starting proteins containing only a few, fairlyconservative, asymmetrical mutations in the CH3 regions.

Thus, in one embodiment, the sequences of said first and second CH3regions contain amino acid substitutions at non-identical positions.

The amino acid substituents may be natural amino acids or unnaturalamino acids. Examples of unnatural amino acids are e.g. disclosed in XieJ and Schultz P. G., Current Opinion in Chemical Biology (2005),9:548-554, and Wang Q. et al., Chemistry & Biology (2009), 16:323-336.

In one embodiment, the amino acids are natural amino acids.

In one embodiment, said first homodimeric protein has no more than oneamino acid substitution in the CH3 region, and the second homodimericprotein has no more than one amino acid substitution in the CH3 regionrelative to the wild-type CH3 regions.

In one embodiment, the first homodimeric protein has an amino acidsubstitution at a position selected from the group consisting of: 366,368, 370, 399, 405, 407 and 409, and said second homodimeric protein hasan amino acid substitution at a position selected from the groupconsisting of: 366, 368, 370, 399, 405, 407 and 409, and wherein saidfirst homodimeric protein and said second homodimeric protein is notsubstituted in the same positions.

In one embodiment, the first homodimeric protein has an amino acidsubstitution at position 366, and said second homodimeric protein has anamino acid substitution at a position selected from the group consistingof: 368, 370, 399, 405, 407 and 409. In one embodiment the amino acid atposition 366 is selected from Arg, Lys, Asn, Gln, Tyr, Glu and Gly.

In one embodiment, the first homodimeric protein has an amino acidsubstitution at position 368, and said second homodimeric protein has anamino acid substitution at a position selected from the group consistingof: 366, 370, 399, 405, 407 and 409.

In one embodiment, the first homodimeric protein has an amino acidsubstitution at position 370, and said second homodimeric protein has anamino acid substitution at a position selected from the group consistingof: 366, 368, 399, 405, 407 and 409.

In one embodiment, the first homodimeric protein has an amino acidsubstitution at position 399, and said second homodimeric protein has anamino acid substitution at a position selected from the group consistingof: 366, 368, 370, 405, 407 and 409.

In one embodiment, the first homodimeric protein has an amino acidsubstitution at position 405, and said second homodimeric protein has anamino acid substitution at a position selected from the group consistingof: 366, 368, 370, 399, 407 and 409.

In one embodiment, the first homodimeric protein has an amino acidsubstitution at position 407, and said second homodimeric protein has anamino acid substitution at a position selected from the group consistingof: 366, 368, 370, 399, 405, and 409.

In one embodiment, the first homodimeric protein has an amino acidsubstitution at position 409, and said second homodimeric protein has anamino acid substitution at a position selected from the group consistingof: 366, 368, 370, 399, 405, and 407.

Accordingly, in one embodiment, the sequences of said first and secondCH3 regions contain asymmetrical mutations, i.e. mutations at differentpositions in the two CH3 regions, e.g. a mutation at position 405 in oneof the CH3 regions and a mutation at position 409 in the other CH3region.

In one embodiment, the first homodimeric protein has an amino acid otherthan Lys, Leu or Met at position 409 and said second homodimeric proteinhas an amino-acid substitution at a position selected from the groupconsisting of: 366, 368, 370, 399, 405 and 407.

In one such embodiment, said first homodimeric protein has an amino acidother than Lys, Leu or Met at position 409 and said second homodimericprotein has an amino acid other than Phe at position 405. In a furtherembodiment hereof, said first homodimeric protein has an amino acidother than Lys, Leu or Met at position 409 and said second homodimericprotein has an amino acid other than Phe, Arg or Gly at position 405.

In another embodiment, said first homodimeric protein comprises a Phe atposition 405 and an amino acid other than Lys, Leu or Met at position409 and said second homodimeric protein comprises an amino acid otherthan Phe at position 405 and a Lys at position 409. In a furtherembodiment hereof, said first homodimeric protein comprises a Phe atposition 405 and an amino acid other than Lys, Leu or Met at position409 and said second homodimeric protein comprises an amino acid otherthan Phe, Arg or Gly at position 405 and a Lys at position 409.

In another embodiment, said first homodimeric protein comprises a Phe atposition 405 and an amino acid other than Lys, Leu or Met at position409 and said second homodimeric protein comprises a Leu at position 405and a Lys at position 409. In a further embodiment hereof, said firsthomodimeric protein comprises a Phe at position 405 and an Arg atposition 409 and said second homodimeric protein comprises an amino acidother than Phe, Arg or Gly at position 405 and a Lys at position 409.

In another embodiment, said first homodimeric protein comprises Phe atposition 405 and an Arg at position 409 and said second homodimericprotein comprises a Leu at position 405 and a Lys at position 409.

In a further embodiment, said first homodimeric protein comprises anamino acid other than Lys, Leu or Met at position 409 and said secondhomodimeric protein comprises a Lys at position 409, a Thr at position370 and a Leu at position 405.

In a further embodiment, said first homodimeric protein comprises an Argat position 409 and said second homodimeric protein comprises a Lys atposition 409, a Thr at position 370 and a Leu at position 405.

In an even further embodiment, said first homodimeric protein comprisesa Lys at position 370, a Phe at position 405 and an Arg at position 409and said second homodimeric protein comprises a Lys at position 409, aThr at position 370 and a Leu at position 405.

In another embodiment, said first homodimeric protein comprises an aminoacid other than Lys, Leu or Met at position 409 and said secondhomodimeric protein comprises a Lys at position 409 and: a) an Ile atposition 350 and a Leu at position 405, or b) a Thr at position 370 anda Leu at position 405.

In another embodiment, said first homodimeric protein comprises an Argat position 409 and said second homodimeric protein comprises a Lys atposition 409 and: a) an Ile at position 350 and a Leu at position 405,or b) a Thr at position 370 and a Leu at position 405.

In another embodiment, said first homodimeric protein comprises a Thr atposition 350, a Lys at position 370, a Phe at position 405 and an Arg atposition 409 and said second homodimeric protein comprises a Lys atposition 409 and: a) an Ile at position 350 and a Leu at position 405,or b) a Thr at position 370 and a Leu at position 405.

In another embodiment, said first homodimeric protein comprises a Thr atposition 350, a Lys at position 370, a Phe at position 405 and an Arg atposition 409 and said second homodimeric protein comprises an Ile atposition 350, a Thr at position 370, a Leu at position 405 and a Lys atposition 409.

In another embodiment, said first homodimeric protein has an amino acidother than Lys, Leu or Met at position 409 and said second homodimericprotein has an amino acid other than Tyr, Asp, Glu, Phe, Lys, Gln, Arg,Ser or Thr at position 407.

In another embodiment, said first homodimeric protein has an amino acidother than Lys, Leu or Met at position 409 and said second homodimericprotein has an Ala, Gly, His, Ile, Leu, Met, Asn, Val or Trp at position407.

In another embodiment, said first homodimeric protein has an amino acidother than Lys, Leu or Met at position 409 and said second homodimericprotein has a Gly, Leu, Met, Asn or Trp at position 407.

In another embodiment, said first homodimeric protein has a Tyr atposition 407 and an amino acid other than Lys, Leu or Met at position409 and said second homodimeric protein has an amino acid other thanTyr, Asp, Glu, Phe, Lys, Gln, Arg, Ser or Thr at position 407 and a Lysat position 409.

In another embodiment, said first homodimeric protein has a Tyr atposition 407 and an amino acid other than Lys, Leu or Met at position409 and said second homodimeric protein has an Ala, Gly, His, Ile, Leu,Met, Asn, Val or Trp at position 407 and a Lys at position 409.

In another embodiment, said first homodimeric protein has a Tyr atposition 407 and an amino acid other than Lys, Leu or Met at position409 and said second homodimeric protein has a Gly, Leu, Met, Asn or Trpat position 407 and a Lys at position 409.

In another embodiment, said first homodimeric protein has a Tyr atposition 407 and an Arg at position 409 and said second homodimericprotein has an amino acid other than Tyr, Asp, Glu, Phe, Lys, Gln, Arg,Ser or Thr at position 407 and a Lys at position 409.

In another embodiment, said first homodimeric protein has a Tyr atposition 407 and an Arg at position 409 and said second homodimericprotein has an Ala, Gly, His, Ile, Leu, Met, Asn, Val or Trp at position407 and a Lys at position 409.

In another embodiment, said first homodimeric protein has a Tyr atposition 407 and an Arg at position 409 and said second homodimericprotein has a Gly, Leu, Met, Asn or Trp at position 407 and a Lys atposition 409.

In one embodiment, the first homodimeric protein has an amino acid otherthan Lys, Leu or Met at position 409, and the second homodimeric proteinhas

-   -   (i) an amino acid other than Phe, Leu and Met at position 368,        or    -   (ii) a Trp at position 370, or    -   (iii) an amino acid other than Asp, Cys, Pro, Glu or Gln at        position 399.

In one embodiment, the first homodimeric protein has an Arg, Ala, His orGly at position 409, and the second homodimeric protein has

-   -   (i) a Lys, Gln, Ala, Asp, Glu, Gly, His, Ile, Asn, Arg, Ser,        Thr, Val, or Trp at position 368, or    -   (ii) a Trp at position 370, or    -   (iii) an Ala, Gly, Ile, Leu, Met, Asn, Ser, Thr, Trp, Phe, His,        Lys, Arg or Tyr at position 399.

In one embodiment, the first homodimeric protein has an Arg at position409, and the second homodimeric protein has

-   -   (i) an Asp, Glu, Gly, Asn, Arg, Ser, Thr, Val, or Trp at        position 368, or    -   (ii) a Trp at position 370, or    -   (iii) a Phe, His, Lys, Arg or Tyr at position 399.

In addition to the above-specified amino-acid substitutions, said firstand second homodimeric protein may contain further amino-acidsubstitutions, deletion or insertions relative to wild-type Fcsequences.

In a further embodiment, said first and second CH3 regions, except forthe specified mutations, comprise the sequence set forth in SEQ ID NO:1(IgG1m(a)):

SEQ ID NO: 1: GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK

In a further embodiment, said first and second CH3 regions, except forthe specified mutations, comprise the sequence set forth in SEQ ID NO:2(IgG1m(f)):

SEQ ID NO: 2: GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK

In a further embodiment, said first and second CH3 regions, except forthe specified mutations, comprise the sequence set forth in SEQ ID NO:3(IgG1m(ax)):

SEQ ID NO: 3: GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEGLHNHYTQKS LSLSPGK

In a further embodiments, the homodimeric proteins provided may be a ratantibody and a mouse antibody, who show preferential pairing, asdescribed by Lindhofer et al. (1995) J Immunol 155:219 (see above), orso-called knob-in-hole variant antibodies, as described in U.S. Pat. No.5,731,168 (see above). In some cases, however, the latter homodimericstarting proteins may be more difficult to produce, because of too weakhomodimeric CH3-CH3 interactions. Thus, the herein described variantshaving mutations at positions 350, 370, 405 and 409, may be preferred.

The sequence of the hinge region of the homodimeric starting proteinsmay vary. However, the resulting heterodimeric protein may be morestable under some circumstances if the hinge region is not IgG4-like,and, preferably is IgG1-like.

Thus, in one embodiment, neither said first nor said second homodimericprotein comprises a Cys-Pro-Ser-Cys sequence in the (core) hinge region.

In a further embodiment, both said first and said second homodimericprotein comprise a Cys-Pro-Pro-Cys sequence in the (core) hinge region.

In many embodiments wherein first and said second homodimeric proteinsare antibodies, said antibodies further comprise a light chain. Asexplained above, said light chains may be different, i.e. differ insequence and each form a functional antigen-binding domain with only oneof the heavy chains. In another embodiment, however, said first andsecond homodimeric proteins are heavy-chain antibodies, which do notneed a light chain for antigen binding, see e.g. Hamers-Casterman (1993)Nature 363:446.

As described above, step c) of the method of the invention comprisesincubating said first protein together with said second protein underreducing conditions sufficient to allow the cysteines in the hingeregion to undergo disulfide-bond isomerisation. Examples of suitableconditions are given herein. The minimal requirements for the cysteinesin the hinge region for undergoing disulfide-bond isomerisation maydiffer depending on the homodimeric starting proteins, in particulardepending on the exact sequence in the hinge region. It is importantthat the respective homodimeric interactions of said first and secondCH3 regions are sufficiently weak to allow cysteines in the hinge regionto undergo disulfide-bond isomerisation under the given conditions.

In one embodiment, the reducing conditions in step c) comprise theaddition of a reducing agent, e.g. a reducing agent selected from thegroup consisting of: 2-mercaptoethylamine (2-MEA), dithiothreitol (DTT),dithioerythritol (DTE), glutathione, tris(2-carboxyethyl)phosphine(TCEP), L-cysteine and beta-mercapto-ethanol, preferably a reducingagent selected from the group consisting of: 2-mercaptoethylamine,dithiothreitol and tris(2-carboxyethyl)phosphine.

In one embodiment, the reducing conditions enabling controlled Fab-armexchange are described in terms of the required redox potential. Thetripeptide glutathione (GSH) is the major low-molecular weight thiol incells and controls thiol-disulphide redox state which is essential fornormal redox signaling in vivo. The dynamics of cellular redox balanceare achieved by maintenance of the thiol-to-disulphide status of reducedGSH and its oxidized form GSSG. The values for the reduction potentialcan be measured as in Rost and Rapoport, Nature 201: 185 (1964) andAslund et al., J. Biol. Chem. 272:30780-30786 (1997). The redoxpotential E_(h), which takes into consideration the stoichiometry of twoGSH oxidized per GSSG is a quantitative measure for the redox state.E_(h) is calculated by the Nernst equation: E_(h)=E_(o)+(RT/nF)ln ([GSSG(ox)]/[GSH (red)]²). Eo is the standard potential for the redox coupleat defined pH, R is the gas constant, T is the absolute temperature, Fis Faraday's constant and n is the number of electrons transferred. Invivo estimates for Eh for the GSH/GSSG couple are in the range of −260to −200 mV (Aw, T., News Physiol. Sci. 18:201-204 (2003)). Terminallydifferentiated cells thereby maintain an Eh in the order of −200 mV,whereas actively proliferating cells maintain a more reduced Eh ofapproximately −260 mV.

The standard redox potential for DTT is −330 mV (Cleland et al.Biochemistry 3: 480-482 (1964)). TCEP has been shown to reduce DTT insolution and therefore has a more negative redox potential than DTT. Theprecise value however has not been reported. Reducing conditionsallowing controlled Fab-arm exchange conditions can therefore bedescribed in terms of a required redox potential Eh, which is optimallybelow the value that is achieved under normal plasma conditions in vivoand that is above the redox potential which reduces antibody disulphidebonds outside those located in the hinge region and involved ininter-heavy chain disulphide bond formation.

Thus, in a further embodiment, step c) is performed under reducingconditions with a redox potential ranging below −50 mV, such as below−150 mV, preferably between −150 and −600 mV, such as between −200 and−500 mV, more preferably between −250 and −450 mV, such as between −250and −400 mV, even more preferably between −260 and −300 mV.

In a further embodiment, step c) comprises incubation for at least 90min at a temperature of at least 20° C. in the presence of at least 25mM 2-mercaptoethylamine or in the presence of at least 0.5 mMdithiothreitol. The incubation may be performed at a pH of from 5 to 8,such as at pH 7.0 or at pH 7.4.

In a further embodiment, step d) comprises restoring the conditions tobecome non-reducing or less reducing, for example by removal of areducing agent, e.g. by desalting.

In some embodiments, the method of the invention yields an antibodyproduct wherein more than 80%, such as more than 90%, e.g. more than95%, such as more than 99% of the antibody molecules are the desiredbispecific antibodies.

The post-production is more flexible and easier to control compared tothe prior art methods based on co-expression.

The post-production nature of making bispecific antibodies byFab-exchange under reducing conditions (such as by addition of 2-MEA) asdisclosed herein makes it a highly suitable strategy for(high-throughput) screening of multiple combinations of specificitiesfor bispecific antibody discovery. In addition, the in vitro process canbe performed in the laboratory which allows greater control, flexibilityand yield of the heterodimeric protein than is allowed by co-expression.An additional advantage of this strategy is that the screening can bedone in the final therapeutic format, precluding the need forengineering upon lead selection.

As explained above, in a further aspect, the method of the invention maybe used for “matrix” screening, i.e. for generating a large number ofdifferent combinations of binding specificities on the basis of two setsof antibodies, one set having identical first CH3 regions and the otherset having identical second CH3 regions, wherein the sequences of saidfirst and second CH3 regions are different and are such that theheterodimeric interaction between said first and second CH3 regions isstronger than each of the homodimeric interactions of said first andsecond CH3 regions.

Thus, in one embodiment the invention relates to a method for theselection of a heterodimeric protein having a desired property, saidmethod comprising the steps of:

-   -   a) providing a first set of homodimeric proteins comprising an        Fc region wherein the homodimeric proteins have identical first        CH3 regions,    -   b) providing a second set of homodimeric proteins comprising an        Fc region wherein the homodimeric proteins have identical second        CH3 regions,    -   wherein the sequences of said first and second CH3 regions are        different and are such that the heterodimeric interaction        between said first and second CH3 regions is stronger than each        of the homodimeric interactions of said first and second CH3        regions,    -   c) incubating combinations of the homodimeric proteins of said        first set and of said second set under reducing conditions        sufficient to allow the cysteines in the hinge region to undergo        disulfide-bond isomerization, thus generating a set of        bispecific antibodies,    -   d) optionally restoring the conditions to non-reducing,    -   e) assaying the resulting set of heterodimeric proteins for a        given desired property, and    -   f) selecting a heterodimeric protein having the desired        property.

In one embodiment, the invention relates to a method for the selectionof a bispecific antibody having a desired property, said methodcomprising the steps of:

-   -   a) providing a first set of homodimeric antibodies comprising        antibodies with different variable regions, wherein said        antibodies of said first set comprise identical first CH3        regions,    -   b) providing a second set of homodimeric antibodies comprising        antibodies with different variable regions or identical variable        regions, wherein said antibodies of said second set comprise        identical second CH3 regions,

wherein the sequences of said first and second CH3 regions are differentand are such that the heterodimeric interaction between said first andsecond CH3 regions is stronger than each of the homodimeric interactionsof said first and second CH3 regions,

-   -   c) incubating combinations of antibodies of said first set and        of said second set under reducing conditions sufficient to allow        the cysteines in the hinge region to undergo disulfide-bond        isomerization, thus generating a set of bispecific antibodies,    -   d) optionally restoring the conditions to non-reducing,    -   e) assaying the resulting set of bispecific antibodies for a        given desired property, and    -   f) selecting a bispecific antibody having the desired property.

In one embodiment, the homodimeric antibodies of the second set havedifferent variable regions.

In one embodiment, the homodimeric antibodies of the second set haveidentical variable regions, but have different amino acid or structuralvariations outside of the antigen binding region.

The two sets can be composed in many different ways as desired. Thus,the two sets may target the same epitope or different epitopes on thesame antigen. The two sets may also target different antigens, and eachset may contain antibodies binding to the same epitope or differentepitopes on the antigen in question. Furthermore, one of the sets orboth sets may each contain antibodies targeting different antigens.

In another embodiment, said desired property is cell killing, celllysis, inhibition of cell proliferation, or binding to cells expressingboth antigen targets. The screening strategy includes two panels ofantibody vectors containing a range of specificities, where one panel iscloned into a backbone that is able to engage in Fab-arm exchange underreducing conditions (such as by addition of 2-MEA) with the backbone ofthe second panel. For example, the first panel is cloned into anIgG1-F405L backbone and the second panel is cloned into a IgG1-K409Rbackbone (for other possible backbone combination see also Examples 19,28, 29, 30, 35, 36, 37, 38, and 39).

Each member of the two panels of antibody vectors is then expressedindividually at small scale. For example, all antibody vectors aretransfected transiently in HEK293 cells and expressed in 2.3 mL culturesin 24-well plates. Alternatively, other suitable (small-scale)production systems known in the art may be used.

The expressed antibodies of the two panels of antibodies are then mixedpair-wise in a matrix-like fashion at equimolar ratios. For example, allindividual antibodies are purified by small-scale protein Achromatography and antibody concentration are measured by absorbance ata wavelength of 280 nm. Other suitable (small-scale) purificationmethods or methods for determining protein concentration known in theart may alternatively be used. In another embodiment, the purificationstep may be left out if down-stream applications are not affected by theexpression medium. Thereafter, the antibody concentrations arenormalized so that a suitable volume contains equimolar amounts of bothantibodies. For example, a panel of 8 antibodies in the F405L backboneis individually mixed with 8 antibodies in the K409R backbone so that 64mixtures of 100 μl contain 80 μg/mL of antibody A (F405L) and 80 μg/mLof antibody B (K409R). Alternatively, if the strategy contains abispecific antibody-specific purification step down-stream, the step tonormalize antibody amounts may be left out.

To the mixtures of antibodies, a suitable amount of reducing agent isadded and incubated for a suitable period of time at a permissivetemperature. For example, to 100 μl containing 80 μg/mL of antibody A(F405L) and 80 μg/mL of antibody B (K409R), 25 μl of 125 mM 2-MEA isadded (final concentration 25 mM 2-MEA) and incubated overnight at 25°C.

The reducing agent is thereupon removed from the mixtures (nowcontaining bispecific antibodies) to promote oxidation of the disulfidebonds and to avoid interference of the reducing agent in the screeningassays. For example, 2-MEA is removed by performing a buffer exchange ofthe 64 mixtures using Zeba Spin 96-well desalting plates (PierceBiotechnology, #89807). Alternatively, other suitable methods to removethe reducing agent known in the art may be used

The bispecific antibodies are then characterized biochemically orfunctionally to identify the lead candidates. For example, the 64bispecific antibodies are assessed for proliferation inhibition ofsuitable cell-lines or binding to suitable cell-lines. Identified leadcandidates will then be produced at larger scale and characterized inmore detail.

Production by Co-Expression

The heterodimeric proteins of the invention may also be obtained byco-expression of constructs encoding the first and second polypeptidesin a single cell.

Thus, in a further aspect, the invention relates to a method forproducing a heterodimeric protein, said method comprising the followingsteps:

a) providing a first nucleic-acid construct encoding a first polypeptidecomprising a first Fc region of an immunoglobulin, said first Fc regioncomprising a first CH3 region,b) providing a second nucleic-acid construct encoding a secondpolypeptide comprising a second Fc region of an immunoglobulin, saidsecond Fc region comprising a first CH3 region,wherein the sequences of said first and second CH3 regions are differentand are such that the heterodimeric interaction between said first andsecond CH3 regions is stronger than each of the homodimeric interactionsof said first and second CH3 regions, and

-   -   wherein said first homodimeric protein has an amino acid other        than Lys, Leu or Met at position 409 and said second homodimeric        protein has an amino-acid substitution at a position selected        from the group consisting of: 366, 368, 370, 399, 405 and 407.    -   and/or    -   wherein the sequences of said first and second CH3 regions are        such that the dissociation constants of homodimeric interactions        of each of the CH3 regions are between 0.01 and 10 micromolar,        such as between 0.05 and 10 micromolar, more preferably between        0.01 and 5, such as between 0.05 and 5 micromolar, even more        preferably between 0.01 and 1 micromolar, such as between 0.05        and 1 micromolar, between 0.01 and 0.5 or between 0.01 and 0.1        when assayed as described in Example 21.        c) co-expressing said first and second nucleic-acid constructs        in a host cell, and        d) obtaining said heterodimeric protein from the cell culture.

Suitable expression vectors, including promoters, enhancers, etc., andsuitable host cells for the production of antibodies are well-known inthe art. Examples of host cells include yeast, bacterial and mammaliancells, such as CHO or HEK cells.

In one embodiment of this method, said first CH3 region has an aminoacid other than Lys, Leu or Met at position 409 and said second CH3region has an amino acid other than Phe at position 405.

and/or

the sequences of said first and second CH3 regions are such that thedissociation constants of homodimeric interactions of each of the CH3regions are between 0.01 and 10 micromolar, such as between 0.05 and 10micromolar, more preferably between 0.01 and 5, such as between 0.05 and5 micromolar, even more preferably between 0.01 and 1 micromolar, suchas between 0.05 and 1 micromolar, between 0.01 and 0.5 or between 0.01and 0.1 when assayed as described in Example 21.

In another embodiment of this method:

-   -   said first CH3 region has an amino acid other than Lys, Leu or        Met at position 409 and said second CH3 region has an amino acid        other than Phe at position 405, such as other than Phe, Arg or        Gly at position 405    -   or    -   said first CH3 region has an amino acid other than Lys, Leu or        Met at position 409 and said second CH3 region has an amino acid        other than Tyr, Asp, Glu, Phe, Lys, Gln, Arg, Ser or Thr at        position 407.

In some embodiments, said first and second polypeptides are full-lengthheavy chains of two antibodies that bind different epitopes (i.e. saidfirst and second nucleic-acid constructs encode full-length heavy chainsof two antibodies that bind different epitopes), and thus theheterodimeric protein is a bispecific antibody. This bispecific antibodycan be a heavy-chain antibody, or said host cell may further express oneor more nucleic-acid constructs encoding a light-chain. If only onelight-chain construct is co-expressed with the heavy chain constructs,then a functional bispecific antibody is only formed if the light chainsequence is such that it can form a functional antigen-binding domainwith each of the heavy chains. If two or more different light-chainconstructs are co-expressed with the heavy chain, multiple products willbe formed.

In further embodiments, the co-expression method according to theinvention comprises any of the further features described under the invitro method above.

In a further aspect, the invention relates to an expression vectorcomprising the first and second nucleic-acid constructs specified hereinabove. In an even further aspect, the invention relates to a host cellcomprising the first and second nucleic-acid constructs specified hereinabove.

Heterodimeric Proteins

In a further aspect, the invention relates to a heterodimeric proteinobtained or obtainable by the method of the invention.

Furthermore, the method of the invention enables the formation ofasymmetrical molecules, molecules with different characteristics on eachof the Fab-arms or on each of the CH3 domains or molecules with distinctmodifications throughout the molecules, e.g. molecules with unnaturalamino acid substitution(s) for conjugation. Such asymmetrical moleculescan be generated in any suitable combinations. This is illustratedfurther below by some non-limiting examples.

Bispecific antibodies can be used to pretarget a target cell ofinterest, including but not limited to, a tumor cell. Pretargeting of atarget cell could be used for imaging studies or for immunotherapeuticpurposes.

In an embodiment of the method of the invention, the first Fab-arm ofthe bispecific molecule binds to a tumor cell, such as a tumor cellsurface protein or tumor cell surface carbohydrate, such as one of thetumor cell surface proteins listed herein and the second Fab-armrecognizes a radioactive effector molecule including but not limited to,a radiolabel coupled or linked (via a chelator) to a peptide or hapten.An example of such radiolabelled peptide is indium-labelleddiethylenetriaminepentaacetic acid (anti-DTPA(In) van Schaijk et al.Clin. Cancer Res. 2005; 11: 7230s-7126s). Another example is usinghapten-labelled colloidal particles such as liposomes, nanoparticles ofpolymeric micelles carrying radionuclides such as for exampletechnetium-99 (Jestin et al. Q J Nuci Med Mol Imaging 2007; 51:51-60).

In another embodiment, a hapten-coupled alternative cytostatic moleculesuch as a toxin is used.

In a further embodiment of the method of the invention, the firstFab-arm of the bispecific molecule is glycosylated at position N297 (EUnumbering) and the second Fab-arm of the bispecific molecules isaglycosylated (nonglycosylated for instance by mutating N297 to Q or Aor E mutation (Bolt S et al., Eur J Immunol 1993, 23:403-411)).Asymmetrical glycosylation in the Fc-region impacts the interaction toFcγ-receptors and has impact on antibody-dependent cell cytotoxicityeffect of the antibody (Ha et al., Glycobiology 2011, Apr. 5) as well asinteraction with other effector function molecules such as C1q.

In another embodiment of the method of the invention, the first Fab-armof the bispecific molecule interacts with FcRn, the neonatal Fc receptor(Roopenian D C, et al. Nat. Rev. Immunol. 2007, 7:715-725) and thesecond Fab-arm is impaired in binding to FcRn by mutation of the FcRninteraction site on the molecules for instance by making a H435Amutation (Shields, R. L., et al, J Biol Chem, 2001, Firan, M., et al,Int Immunol, 2001).

In another embodiment of the method of the invention, the first Fab-armof the bispecific molecule interacts with staphylococcal protein A(protein A, Deisenhofer et al, Biochemistry 20, 2361-2370 (1981) andstreptococcal protein G (protein G, Derrick et al., Nature 359, 752-754(1992), often used for purification of antibodies, and the secondFab-arm of bispecific molecules is impaired in the interaction withprotein A of G. As a result, removal of residual amounts of homodimerwith impaired protein A or G binding after the exchange into heterodimeris easily obtained by purification of the bispecific molecule withprotein A or G.

In another embodiment, the binding to either Fcγ-receptors or FcRn isimproved or decreased on one of the two Fab-arms of the bispecificmolecule.

In another embodiment, the binding to C1q is improved or decreased onone of the two Fab-arms of the bispecific molecule.

In another embodiment, the protein has been engineered to enhancecomplement activation on one or both of the two Fab-arms of themolecule.

In another embodiment, each of the Fab-arms present in the bispecificmolecule is derived from a different IgG subclass.

In another embodiment, each of the Fab-arms present in the bispecificmolecule carry different allotypic mutations (Jefferis & Lefranc, 2009,MABs 1:332-8).

In another embodiment, another category of asymmetric immunotherapeuticmolecules is generated by replacement of the Fab of one of the Fab-armsof the bispecific molecule by an immuno active, stimulating orinhibiting cytokine. Non-limiting examples of such cytokines are IL-2,IFN-α, IFN-β, IFN-γ, TNF-α, G-CSF, GM-CSF, IL-10, IL-4, IL-6, IL-13.Alternatively, a (growth) factor or hormone stimulating or inhibitionagent is included in the molecules.

In another embodiment, a Fab of one of the Fab-arms is replaced by alytic peptide, i. e. peptides that are able to lyse tumor cells,bacteria, fungi etc, including but not limited to antimicrobial peptideslike magainin, mellitin, cecropin, KLAKKLAK and variants thereof(Schweizer et al. Eur. J. Pharmacology 2009; 625: 190-194, Javadpour, J.Med. Chem., 1996, 39: 3107-3113, Marks et al, Cancer Res 2005;65:2373-2377, Rege et al, Cancer Res. 2007; 67:6368-6375) or cationiclytic peptides (CLYP technology, US2009/0269341).

In another embodiment, one or both of the Fabs on the Fab arms isreplaced by receptors for cytokines and/or growth factors, creating socalled decoy receptors, of which Enbrel® (etanercept) targeting TNF-αand VEGF-trap, targeting VEGF, are well-known examples. Combining thesetwo decoy receptors into one molecule showed superior activity over thesingle decoy receptors (Jung, J. Biol. Chem. 2011; 286:14410-14418).

In another embodiment, another category of asymmetric immunotherapeuticmolecules is generated by fusion of immuno-active, -stimulating orinhibiting cytokines to the N-terminus or C-terminus of one, or both, ofthe Fab-arms present in the bispecific molecules. This may positivelyimpact anti-tumor activity of the bispecific molecule. Examples of suchmolecules, however not limited to the list below, are IL-2 (Fournier etal., 2011, Int. J. Oncology, doi: 10.3892/ijo.2011.976), IFN-α, IFN-β orIFN-γ (Huan et al., 2007; J. Immunol. 179:6881-6888, Rossie et al.,2009; Blood 114: 3864-3871), TNF-α. Alternatively, N-terminal orC-terminal fusion of cytokines, such as for example G-CSF, GM-CSF,IL-10, IL-4, IL-6, or IL-13 may positively impact the bispecificantibody molecule effector function. Alternatively a (growth) factor orhormone stimulating or inhibition agent is included in the molecules onthe N-terminus or C-terminus.

In another embodiment, N-terminal or C-terminal fusion of a lyticpeptide, such as for example antimicrobial peptides like magainin,mellitin, cecropin, KLAKKLAK and variants thereof (Schweizer et al. Eur.J. Pharmacology 2009; 625: 190-194, Javadpour, J. Med. Chem., 1996, 39:3107-3113, Marks et al, Cancer Res 2005; 65:2373-2377, Rege et al,Cancer Res. 2007; 67:6368-6375) or cationic lytic peptides (CLYPtechnology, US2009/0269341) on one or both of the Fab-ams may enhancethe activity of the molecule.

In another embodiment, another category of asymmetric immunotherapeuticmolecules is monovalent antibodies, molecules which interact with oneFab-arm to the target of choice. In such molecule one of the Fab-armspresent in the bispecific molecule is directed against the targetmolecule of choice, the second Fab-arm of the molecule does not carry aFab or has a non-binding/non-functional Fab such as described for MetMab(Genentech; WO 96/38557). Alternatively, monomeric Fc-fusion proteinssuch as those described for Factor VIII and IX (Peters et al., Blood2010; 115: 2057-2064) may be generated.

Alternatively, combinations of any of the above mentioned asymmetricalmolecules may be generated by the method of the invention.

In an even further aspect, the invention relates to a heterodimericprotein comprising a first polypeptide comprising a first Fc region ofan immunoglobulin, said first Fc region comprising a first CH3 region,and a second polypeptide comprising a second Fc region of animmunoglobulin, said second Fc region comprising a second CH3 region,wherein the sequences of said first and second CH3 regions are differentand are such that the heterodimeric interaction between said first andsecond CH3 regions is stronger than each of the homodimeric interactionsof said first and second CH3 regions, and

-   -   wherein said first homodimeric protein has an amino acid other        than Lys, Leu or Met at position 409 and said second homodimeric        protein has an amino-acid substitution at a position selected        from the group consisting of: 366, 368, 370, 399, 405 and 407    -   and/or    -   wherein the sequences of said first and second CH3 regions are        such that the dissociation constants of homodimeric interactions        of each of the CH3 regions are between 0.01 and 10 micromolar,        such as between 0.05 and 10 micromolar, more preferably between        0.01 and 5, such as between 0.05 and 5 micromolar, even more        preferably between 0.01 and 1 micromolar, such as between 0.05        and 1 micromolar, between 0.01 and 0.5 or between 0.01 and 0.1        when assayed as described in Example 21.        In one embodiment, said first CH3 region has an amino acid other        than Lys, Leu or Met at position 409 and said second CH3 region        has an amino acid other than Phe at position 405    -   and/or    -   the sequences of said first and second CH3 regions are such that        the dissociation constants of homodimeric interactions of each        of the CH3 regions are between 0.01 and 10 micromolar, such as        between 0.05 and 10 micromolar, more preferably between 0.01 and        5, such as between 0.05 and 5 micromolar, even more preferably        between 0.01 and 1 micromolar, such as between 0.05 and 1        micromolar, between 0.01 and 0.5 or between 0.01 and 0.1 when        assayed as described in Example 21.        In a further embodiment of the heterodimeric protein    -   said first CH3 region has an amino acid other than Lys, Leu or        Met at position 409 and said second CH3 region has an amino acid        other than Phe at position 405, such as other than Phe, Arg or        Gly, at position 405    -   or    -   said first CH3 region has an amino acid other than Lys, Leu or        Met at position 409 and said second CH3 region has an amino acid        other than Tyr, Asp, Glu, Phe, Lys, Gln, Arg, Ser or Thr at        position 407.

In further embodiments, the heterodimeric protein according to theinvention comprises any of the further features described above for themethods of production.

Thus, in a further embodiment of the heterodimeric protein of theinvention, said first polypeptide is a full-length heavy chain of anantibody, preferably a human antibody.

In another embodiment of the heterodimeric protein of the invention,said second polypeptide is a full-length heavy chain of an antibody,preferably a human antibody.

In a further embodiment of the heterodimeric protein of the invention,said first and second polypeptides are both full-length heavy chains oftwo antibodies, preferably both human antibodies that bind differentepitopes, and thus the resulting heterodimeric protein is a bispecificantibody. This bispecific antibody can be a heavy-chain antibody, or anantibody which in addition to the heavy chains comprises two full-lengthlight chains, which may be identical or different.

In a further embodiment of the heterodimeric protein of the invention,the Fc region of the first polypeptide is of an isotype selected fromthe group consisting of IgG1, IgG2, IgG3 and IgG4 (except for thespecified mutations) and the Fc region of the second polypeptide is ofan isotype selected from the group consisting of IgG1, IgG2, IgG3 andIgG4 (except for the specified mutations).

In a further embodiment of the heterodimeric protein of the invention,the Fc regions of both said first and said second polypeptides are ofthe IgG1 isotype.

In a further embodiment of the heterodimeric protein of the invention,one of the Fc regions of said polypeptides is of the IgG1 isotype andthe other of the IgG4 isotype.

In a further embodiment of the heterodimeric protein of the invention,the increased strength of the heterodimeric interaction as compared toeach of the homodimeric interactions is due to CH3 modifications otherthan the introduction of covalent bonds, cysteine residues or chargedresidues.

In a further embodiment of the heterodimeric protein of the invention,the heterodimeric interaction between said first and second polypeptidesin the heterodimeric protein is such that no Fab-arm exchange can occurat 0.5 mM GSH under the conditions described in Example 13.

In a further embodiment of the heterodimeric protein of the invention,the heterodimeric interaction between said first and second polypeptidesin the resulting heterodimeric protein is such that no Fab-arm exchangeoccurs in vivo in mice under the conditions described in Example 14.

In a further embodiment of the heterodimeric protein of the invention,said first CH3 region comprises a Phe at position 405 and an amino acidother than Lys, Leu or Met at position 409 and said second CH3 regioncomprises an amino acid other than Phe at position 405 and a Lys atposition 409.

In a further embodiment of the heterodimeric protein of the invention,said first CH3 region comprises a Phe at position 405 and an amino acidother than Lys, Leu or Met at position 409 and said second CH3 regioncomprises a Leu at position 405 and a Lys at position 409.

In a further embodiment of the heterodimeric protein of the invention,said first CH3 region comprises Phe at position 405 and an Arg atposition 409 and said second CH3 region comprises a Leu at position 405and a Lys at position 409.

In a further embodiment of the heterodimeric protein of the invention,said first CH3 region comprises an amino acid other than Lys, Leu or Metat position 409 and said second CH3 region comprises a Lys at position409 and: a) an Ile at position 350 and a Leu at position 405, or b) aThr at position 370 and a Leu at position 405.

In a further embodiment of the heterodimeric protein of the invention,said first CH3 region comprises an Arg at position 409 and said secondCH3 region comprises a Lys at position 409 and: a) an Ile at position350 and a Leu at position 405, or b) a Thr at position 370 and a Leu atposition 405.

In a further embodiment of the heterodimeric protein of the invention,said first CH3 region comprises a Thr at position 350, a Lys at position370, a Phe at position 405 and an Arg at position 409 and said secondCH3 region comprises a Lys at position 409 and: a) an Ile at position350 and a Leu at position 405, or b) a Thr at position 370 and a Leu atposition 405.

In a further embodiment of the heterodimeric protein of the invention,said first CH3 region comprises a Thr at position 350, a Lys at position370, a Phe at position 405 and an Arg at position 409 and said secondCH3 region comprises an Ile at position 350, a Thr at position 370, aLeu at position 405 and a Lys at position 409.

In a further embodiment of the heterodimeric protein of the invention,neither said first nor said second polypeptide comprises aCys-Pro-Ser-Cys sequence in the hinge region.

In a further embodiment of the heterodimeric protein of the invention,both said first and said second polypeptide comprise a Cys-Pro-Pro-Cyssequence in the hinge region.

In a further embodiment of the heterodimeric protein of the invention,said first and/or said second polypeptide comprises a mutation removingthe acceptor site for Asn-linked glycosylation.

Target Antigens

As explained above, in an important embodiment of the invention, theheterodimeric protein is a bispecific antibody comprising two variableregions that differ in binding specificity, i.e. bind differentepitopes.

In principle, any combination of specificities is possible. As mentionedabove, bispecific antibodies can potentially be used to overcome some ofthe limitations of monospecific antibodies. One possible limitation of amonospecific antibody is a lack of specificity for the desired targetcells due to expression of the target antigen on other cell typestowards which no antibody binding is desired. For example, a targetantigen overexpressed on tumor cells may also be expressed in healthytissues which could result in undesired side-effects upon treatment withan antibody directed against that antigen. A bispecific antibody havinga further specificity against a protein which is exclusively expressedon the target cell type could potentially improve specific binding totumor cells.

Thus, in one embodiment of the invention, said first and second epitopesare located on the same cell, e.g. a tumor cell. Suitable targets ontumor cells include, but are not limited to, the following: erbB1(EGFR), erbB2 (HER2), erbB3, erbB4, MUC-1, CD19, CD20, CD4, CD38, CD138,CXCR5, c-Met, HERV-envelop protein, periostin, Bigh3, SPARC, BCR, CD79,CD37, EGFrvIII, L1-CAM, AXL, Tissue Factor (TF), CD74, EpCAM and MRP3.Possible combinations of tumor cell targets include, but are not limitedto: erbB1+erbB2, erbB2+erbB3, erbB1+erbB3, CD19+CD20, CD38+CD34,CD4+CXCR5, CD38+RANKL, CD38+CXCR4, CD20+CXCR4, CD20+CCR7, CD20+CXCR5,CD20+RANKL, erbB2+AXL, erbB1+cMet, erbB2+c-Met, erbB2+EpCAM, c-Met+AXL,c-Met+TF, CD38+CD20, CD38+CD138.

In a further embodiment, said first and second epitopes may be locatedon the same target antigen, wherein the location of the two epitopes onthe target antigen is such that binding of an antibody to one epitopedoes not interfere with antibody binding to the other epitope. In afurther embodiment hereof, said first and second homodimeric proteinsare antibodies that bind to two different epitopes located on the sametarget antigen, but have a different mode-of-action for killing thetarget cell, e.g. a tumor cell. For example, in one embodiment, thetarget antigen is erbB2 (HER2) and the bispecific antibody combines thepertuzumab and trastuzumab antigen-binding sites. In another embodiment,the target antigen is erbB1 (EGFr) and the bispecific antibody combinesthe zalutumumab and nimotuzumab antigen-binding sites.

Bispecific antibodies can also be used as mediators to retarget effectormechanisms to disease-associated tissues, e.g. tumors. Thus, in afurther embodiment, said first or said second epitope is located on atumor cell, such as a tumor cell protein or tumor cell carbohydrate, andthe other epitope is located on an effector cell.

In one embodiment, the effector cell is a T cell.

Possible targets on effector cells include the following: FcgammaRI(CD64): expressed on monocytes and macrophages and activatedneutrophils; FcgammaRIII (CD16): expressed on natural killer andmacrophages; CD3: expressed on circulating T cells; CD89: expressed onPMN (polymorphonuclear neutrophils), eosinophils, monocytes andmacrophages; CD32a: expressed on macrophages, neutrophils, eosinophils;FcεRI expressed on basophils and mast cells. In one embodiment theepitope is located on CD3 expressed on T cells.

In another embodiment, the first antibody has binding specificity for apathogenic microorganism and the second antibody has binding specificityfor an effector cell protein, such as CD3, CD4, CD8, CD40, CD25, CD28,CD16, CD89, CD32, CD64, FcεRI or CD1.

Furthermore, bispecific antibodies can be used to target achemotherapeutic agent more specifically to the cells on which the agentshould act. Thus, in one embodiment, one of the homodimeric proteins isan antibody which recognizes a small molecule or peptide, or is able toform a covalent bond with such a molecule, e.g. according to theprinciple described in Rader et al, (2003) PNAS 100:5396. In a furtherembodiment of the method of the invention, the first antibody hasbinding specificity for (i.e. binds to an epitope on) a tumor cell ortumor cell surface protein, such as erbB1, erbB2, erbB3, erbB4,EGFR3vIII, CEA, MUC-1, CD19, CD20, CD4, CD38, EPCAM, c-Met, AXL, L1-CAM,Tissue Factor, CD74 or CXCR5 and the second antibody has a bindingspecificity for a chemotherapeutic agent, such as a toxin (including aradiolabelled peptide), a drug or a prodrug.

Bispecific antibodies may also be used to target a vesicle, e.g. anelectron dense vesicles, or minicell containing a toxin, drug or prodrugto a tumor. See e.g. MacDiarmid et al. (2009) Nature Biotech 27:643.Minicells are achromosomal cells that are products of aberrant celldivision which do not contain chromosomal DNA. Thus, in anotherembodiment, wherein said first or said second epitope is located on atumor cell, such as a tumor cell protein or tumor cell carbohydrate, andthe other epitope is located on an electron dense vesicle or minicell.

Furthermore, serum half-life of an antibody may be altered by includingin a bispecific antibody a binding specificity for a serum protein. Forinstance, serum half-life may be prolonged by including in a bispecificantibody, a binding specificity for serum albumin. Thus, in a furtherembodiment of the method of the invention, the first antibody hasbinding specificity for a tumor cell or tumor cell protein, such aserbB1 (EGFR), erbB2 (HER2), erbB3, erbB4, MUC-1, CD19, CD20, CD4, CD38,CD138, CXCR5, c-Met, HERV-envelope protein, periostin, Bigh3, SPARC,BCR, CD79, CD37, EGFrvIII, L1-CAM, AXL, Tissue Factor (TF), CD74, EpCAMor MRP3, CEA and the second antibody has a binding specificity for ablood protein, such as serum albumin. A second binding specificity canalso be used to target an antibody to a specific tissue, such as thecentral nervous system or brain (across the blood brain barrier). Thus,in a further embodiment of the method of the invention, the firstantibody has binding specificity for a brain-specific target, such asamyloid-beta (e.g. for treatment of Alzheimer's disease), Her-2 (e.g.for treatment of breast cancer metastases in brain), EGFr (e.g. fortreatment of primary brain cancer), Nogo A (e.g. for treatment of braininjury), TRAIL (e.g. for treatment of HIV), alpha-synuclein (e.g. fortreatment of Parkinson), Htt (e.g. for treatment of Huntington), a prion(e.g. for treatment of mad cow disease), a West Nile virus protein, andthe second antibody has a binding specificity for a blood brain barrierprotein, such as transferrin receptor (TfR), insulin receptor,melanotransferrin receptor (MTfR), lactoferrin receptor (LfR),Apolipoprotein E receptor 2 (ApoER2), LDL-receptor-related protein 1 and2 (LRP1 and LRP2), receptor for advanced glycosylation end-products(RAGE), diphtheria toxin-receptor=heparin-binding epidermal growthfactor-like growth factor (DTR=HB-EGF), gp190 (Abbott et al,Neurobiology of Disease 37 (2010) 13-25).

A binding specificity for a blood brain barrier protein can also be usedto target another, non-antibody, molecule, to a specific tissue, such asthe central nervous system or brain (across the blood brain barrier).Thus, in a further embodiment, one of the homodimeric proteins is afull-length antibody having a binding specificity for a blood brainbarrier protein (such as TfR, insulin receptor, MTfR, LfR, ApoER2, LRP1,LRP2, RAGE, DTR (=HB-EGF) or gp190) and the other homodimeric protein isan Fc region linked at the N- or C-terminus to another protein, such asa cytokine, a soluble receptor or other protein, e.g. VIP (vasoactiveintestinal peptide), BDNF (brain-derived neurotrophic factor), FGF(fibroblast growth factor), multiple FGFs, EGF (epidermal growthfactor), PNA (peptide nucleic acid), NGF (Nerve growth factor),Neurotrophin (NT)-3, NT-4/5, glial derived neurotrophic factor, ciliaryneurotrophic factor, neurturin, neuregulins, interleukins, transforminggrowth factor (TGF)-alpha, TGF-beta, erythropoietin, hepatocyte growthfactor, platelet derived growth factor, artemin, persephin, netrins,cardiotrophin-1, stem cell factor, midkine, pleiotrophin, bonemorphogenic proteins, saposins, semaphorins, leukocyte inhibitoryfactor, alpha-L-iduronidase, iduronate-2-sulfatase,N-acetyl-galactosamine-6-sulfatase, arylsulphatase B, acidalpha-glucosidase, or sphingomyelinase (Pardridge, Bioparmaceutical drugtargeting to the brain, Journal of Drug Targeting 2010, 1-11; Pardridge,Re-engineering Biopharmaceuticals for delivery to brain with molecularTrojan horses. Bioconjugate Chemistry 2008, 19: 1327-1338.

Moreover, a second binding specificity can be used to target bloodclotting factors to a particular desired site of action. For example, abispecific antibody having a first binding specificity for a tumor celland a second binding specificity for a blood clotting factor coulddirect blood clotting to a tumor, and thus stop tumor growth. Thus, in afurther embodiment of the method of the invention, the first antibodyhas binding specificity for a tumor cell or tumor cell protein, such aserbB1, erbB2, erbB3, erbB4, MUC-1, CD19, CD20, CD4 or CXCR5 and thesecond antibody has a binding specificity for a protein involved inblood clotting, such as tissue factor.

Further particularly interesting binding specificity combinationsinclude: CD3+HER2, CD3+CD20, IL-12+IL18, IL-1a+IL-1b, VEGF+EGFR,EpCAM+CD3, GD2+CD3, GD3+CD3, HER2+CD64, EGFR+CD64, CD30+CD16, NG2+CD28,HER2+HER3, CD20+CD28, HER2+CD16, Bcl2+CD3, CD19+CD3, CEA+CD3, EGFR+CD3,IgE+CD3, EphA2+CD3, CD33+CD3, MCSP+CD3, PSMA+CD3, TF+CD3, CD19+CD16,CD19+CD16a, CD30+CD16a, CEA+HSG, CD20+HSG, MUC1+HSG, CD20+CD22,HLA-DR+CD79, PDGFR+VEGF, IL17a+IL23, CD32b+CD25, CD20+CD38, HER2+AXL,CD89+HLA class II, CD38+CD138, TF+cMet, Her2+EpCAM, HER2+HER2,EGFR+EGFR, EGFR+c-Met, c-Met+non-binding arm and combinations ofG-protein coupled receptors.

In a further embodiment, the bispecific antibodies according to theinvention may be used to clear pathogens, pathogenic autoantibodies orharmful compounds such as venoms and toxins from the circulation bytargeting to erythrocytes essentially as described in Taylor et al. J.Immunol. 158:842-850 (1997) and Taylor and Ferguson, J. Hematother.4:357-362, 1995. Said first epitope is located on an erythrocyte (redblood cell) protein including, but not limited to, the erythrocytecomplement receptor 1 and said second epitope is located on the compoundor organism to be targeted for clearance.

In a further embodiment, the second Fab-arm comprises a fusion proteinrepresenting an autoantigen or a conjugation site to attach anautoantigen such as dsDNA. Targeting of pathogens, autoantibodies orharmful compounds by the bispecific antibodies of the invention followedby erythrocyte-mediated clearance may thus have therapeutic utility inthe treatment of various diseases and syndromes.

Conjugation

In further embodiments of the invention, the first and/or secondhomodimeric protein is linked to a compound selected from the groupconsisting of: a toxin (including a radioisotope) a prodrug or a drug.Such compound may make killing of target cells more effective, e.g. incancer therapy. The resulting heterodimeric protein is thus animmunoconjugate. The compound may alternatively be coupled to theresulting heterodimeric protein, i.e. after the Fab-arm exchange hastaken place.

Suitable compounds for forming immunoconjugates of the present inventioninclude taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine,mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin,doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone,mithramycin, actinomycin D, 1-dehydro-testosterone, glucocorticoids,procaine, tetracaine, lidocaine, propranolol, and puromycin,antimetabolites (such as methotrexate, 6-mercaptopurine, 6-thioguanine,cytarabine, fludarabin, 5-fluorouracil, decarbazine, hydroxyurea,asparaginase, gemcitabine, cladribine), alkylating agents (such asmechlorethamine, thioepa, chlorambucil, melphalan, carmustine (BSNU),lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol,streptozotocin, dacarbazine (DTIC), procarbazine, mitomycin C, cisplatinand other platinum derivatives, such as carboplatin), antibiotics (suchas dactinomycin (formerly actinomycin), bleomycin, daunorubicin(formerly daunomycin), doxorubicin, idarubicin, mithramycin, mitomycin,mitoxantrone, plicamycin, anthramycin (AMC)), diphtheria toxin andrelated molecules (such as diphtheria A chain and active fragmentsthereof and hybrid molecules), ricin toxin (such as ricin A or adeglycosylated ricin A chain toxin), cholera toxin, a Shiga-like toxin(SLT-I, SLT-II, SLT-IIV), LT toxin, C3 toxin, Shiga toxin, pertussistoxin, tetanus toxin, soybean Bowman-Birk protease inhibitor,Pseudomonas exotoxin, alorin, saporin, modeccin, gelanin, abrin A chain,modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthinproteins, Phytolacca americana proteins (PAPI, PAPII, and PAP-S),momordica charantia inhibitor, curcin, crotin, sapaonaria officinalisinhibitor, gelonin, mitogellin, restrictocin, phenomycin, and enomycintoxins. Other suitable conjugated molecules include ribonuclease(RNase), DNase I, Staphylococcal enterotoxin-A, pokeweed antiviralprotein, diphtherin toxin, Pseudomonas endotoxin, Maytansinoids,Auristatins (MMAE, MMAF), Calicheamicins and Duocarmycin analogs (Ducryand Stump, Bioconjugate Chem. 2010, 21: 5-13), Dolostatin-10,Dolostatin-15, Irinotecan or its active metabolite SN38,pyrrolobenzodiazepines (PBD's).

In a further embodiment of the invention, the first and/or secondhomodimeric protein is linked to an alpha emitter, including but notlimited to Thorium-227, Radium-223, Bismuth-212, and Actinium-225.

In a further embodiment of the invention, the first and/or secondhomodimeric protein is linked to a beta emitting radionuclide, includingbut not limited to Iodium-313, Yttrium-90, Fluorine-18, Rhenium-186,Gallium-68, Technetium-99, Indium-111, and Lutetium-177.

In another embodiment, the compound to be conjugated comprises a nucleicacid or nucleic acid-associated molecule. In one such facet of thepresent invention, the conjugated nucleic acid is a cytotoxicribonuclease, an antisense nucleic acid, an inhibitory RNA molecule(e.g., a siRNA molecule) or an immunostimulatory nucleic acid (e.g., animmunostimulatory CpG motif-containing DNA molecule).

Any method known in the art for conjugating may be employed, includingthe methods described by Hunter et al., Nature 144, 945 (1962), David etal., Biochemistry 13, 1014 (1974), Pain et al., J. Immunol. Meth. 40,219 (1981) and Nygren, J. Histochem. and Cytochem. 30, 407 (1982).Conjugates may be produced by chemically conjugating the other moiety tothe N-terminal side or C-terminal side of the protein (see, e.g.,Antibody Engineering Handbook, edited by Osamu Kanemitsu, published byChijin Shokan (1994)). Such conjugated antibody derivatives may also begenerated by conjugation at internal residues or sugars, whereappropriate. The agents may be coupled either directly or indirectly toa protein of the present invention. One example of indirect coupling ofa second agent is coupling by a spacer moiety. Linking technologies fordrug-conjugates have recently been summarized by Ducry and Stump (2010)Bioconjugate Chem. 21: 5.

Compositions and Uses

In a further main aspect, the invention relates to a pharmaceuticalcomposition comprising a heterodimeric protein according to theinvention as described herein and a pharmaceutically-acceptable carrier.

The pharmaceutical compositions may be formulated in accordance withconventional techniques such as those disclosed in Remington: TheScience and Practice of Pharmacy, 19th Edition, Gennaro, Ed., MackPublishing Co., Easton, Pa., 1995. A pharmaceutical composition of thepresent invention may e.g. include diluents, fillers, salts, buffers,detergents (e. g., a nonionic detergent, such as Tween-20 or Tween-80),stabilizers (e. g., sugars or protein-free amino acids), preservatives,tissue fixatives, solubilizers, and/or other materials suitable forinclusion in a pharmaceutical composition.

Pharmaceutically acceptable carriers include any and all suitablesolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonicity agents, antioxidants and absorption delaying agents,and the like that are physiologically compatible with a compound of thepresent invention. Examples of suitable aqueous and nonaqueous carrierswhich may be employed in the pharmaceutical compositions of the presentinvention include water, saline, phosphate buffered saline, ethanol,dextrose, polyols (such as glycerol, propylene glycol, polyethyleneglycol). Pharmaceutically acceptable carriers include sterile aqueoussolutions or dispersions and sterile powders for the extemporaneouspreparation of sterile injectable solutions or dispersion. Properfluidity may be maintained, for example, by the use of coatingmaterials, such as lecithin, by the maintenance of the required particlesize in the case of dispersions, and by the use of surfactants.

Pharmaceutical compositions of the present invention may also comprisepharmaceutically acceptable antioxidants for instance (1) water solubleantioxidants, such as ascorbic acid, cysteine hydrochloride, sodiumbisulfate, sodium metabisulfite, sodium sulfite and the like; (2)oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole, butylated hydroxytoluene, lecithin, propyl gallate,alpha-tocopherol, and the like; and (3) metal chelating agents, such ascitric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaricacid, phosphoric acid, and the like.

Pharmaceutical compositions of the present invention may also compriseisotonicity agents, such as sugars, polyalcohols, such as mannitol,sorbitol, glycerol or sodium chloride in the compositions.

The pharmaceutical compositions of the present invention may alsocontain one or more adjuvants appropriate for the chosen route ofadministration such as preservatives, wetting agents, emulsifyingagents, dispersing agents, preservatives or buffers, which may enhancethe shelf life or effectiveness of the pharmaceutical composition. Thecompounds of the present invention may be prepared with carriers thatwill protect the compound against rapid release, such as a controlledrelease formulation, including implants, transdermal patches, andmicroencapsulated delivery systems. Such carriers may include gelatin,glyceryl monostearate, glyceryl distearate, biodegradable, biocompatiblepolymers such as ethylene vinyl acetate, polyanhydrides, polyglycolicacid, collagen, polyorthoesters, and polylactic acid alone or with awax, or other materials well known in the art. Methods for thepreparation of such formulations are generally known to those skilled inthe art.

Sterile injectable solutions may be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients e.g. as enumerated above, as required,followed by sterilization microfiltration.

The actual dosage levels of the active ingredients in the pharmaceuticalcompositions may be varied so as to obtain an amount of the activeingredient which is effective to achieve the desired therapeuticresponse for a particular patient, composition, and mode ofadministration, without being toxic to the patient. The selected dosagelevel will depend upon a variety of pharmacokinetic factors includingthe activity of the particular compositions of the present inventionemployed, the route of administration, the time of administration, therate of excretion of the particular compound being employed, theduration of the treatment, other drugs, compounds and/or materials usedin combination with the particular compositions employed, the age, sex,weight, condition, general health and prior medical history of thepatient being treated, and like factors well known in the medical arts.

The pharmaceutical composition may be administered by any suitable routeand mode. In one embodiment, a pharmaceutical composition of the presentinvention is administered parenterally. “administered parenterally” asused herein means modes of administration other than enteral and topicaladministration, usually by injection, and include epidermal,intravenous, intramuscular, intraarterial, intrathecal, intracapsular,intraorbital, intracardiac, intradermal, intraperitoneal,intratendinous, transtracheal, subcutaneous, subcuticular,intraarticular, subcapsular, subarachnoid, intraspinal, intracranial,intrathoracic, epidural and intrasternal injection and infusion.

In one embodiment that pharmaceutical composition is administered byintravenous or subcutaneous injection or infusion.

In a main aspect, the invention relates to a heterodimeric proteinaccording to the invention, such as a bispecific antibody according tothe invention, for use as a medicament. The heterodimeric protein of theinvention may be used for a number of purposes. In particular, asexplained above the heterodimeric proteins of the invention may be usedfor the treatment of various forms of cancer, including metastaticcancer and refractory cancer.

Thus, in one aspect, the invention relates to a method for inhibitinggrowth and/or proliferation of and/or for killing of a tumor cellcomprising administration of a heterodimeric protein according to theinvention as described herein to an individual in need thereof.

In another embodiment the heterodimeric proteins of the invention areused for the treatment of immune and autoimmune diseases, inflammatorydiseases, infectious diseases, cardiovascular diseases, CNS andmusculo-skeletal diseases.

Dosage regimens in the above methods of treatment and uses are adjustedto provide the optimum desired response (e.g., a therapeutic response).For example, a single bolus may be administered, several divided dosesmay be administered over time or the dose may be proportionally reducedor increased as indicated by the exigencies of the therapeuticsituation.

The efficient dosages and the dosage regimens for the heterodimericproteins depend on the disease or condition to be treated and may bedetermined by the persons skilled in the art. An exemplary, non-limitingrange for a therapeutically effective amount of a bispecific antibody ofthe present invention is about 0.1-100 mg/kg, such as about 0.1-50mg/kg, for example about 0.1-20 mg/kg, such as about 0.1-10 mg/kg, forinstance about 0.5, about such as 0.3, about 1, about 3, about 5, orabout 8 mg/kg.

A physician or veterinarian having ordinary skill in the art may readilydetermine and prescribe the effective amount of the pharmaceuticalcomposition required. For example, the physician or veterinarian couldstart doses of the heterodimeric protein employed in the pharmaceuticalcomposition at levels lower than that required in order to achieve thedesired therapeutic effect and gradually increase the dosage until thedesired effect is achieved. In general, a suitable daily dose of acomposition of the present invention will be that amount of the compoundwhich is the lowest dose effective to produce a therapeutic effect.Administration may e.g. be parenteral, such as intravenous,intramuscular or subcutaneous.

A heterodimeric protein of the invention may also be administeredprophylactically in order to reduce the risk of developing disease, suchas cancer, delay the onset of the occurrence of an event in diseaseprogression, and/or reduce the risk of recurrence when a disease, suchas cancer is in remission.

Heterodimeric proteins, such as bispecific antibodies, of the presentinvention may also be administered in combination therapy, i.e.,combined with other therapeutic agents relevant for the disease orcondition to be treated. Accordingly, in one embodiment, theHeterodimeric-protein-containing medicament is for combination with oneor more further therapeutic agents, such as a cytotoxic,chemotherapeutic or anti-angiogenic agents. Such combined administrationmay be simultaneous, separate or sequential. In a further embodiment,the present invention provides a method for treating or preventingdisease, such as cancer, which method comprises administration to asubject in need thereof of a therapeutically effective amount of aheterodimeric protein, such as a bispecific antibody of the presentinvention, in combination with radiotherapy and/or surgery.

Heterodimeric proteins, such as bispecific antibodies, of the presentinvention may also be used for diagnostic purposes.

EXAMPLES Example 1 Expression Vectors for the Expression of HumanIgG1-2F8 and IgG1-7D8

The VH and VL coding regions of HuMab 2F8 (WO 02/100348) and HuMab 7D8(WO 04/035607) were cloned in the expression vector pConG1f (containingthe genomic sequence of the human IgG1f allotype constant region (LonzaBiologics)) for the production of the human IgG1 heavy chain andpConKappa (containing the human kappa light chain constant region, LonzaBiologics) for the production of the kappa light chain. For IgG4antibodies the VH regions were inserted in the pTomG4 vector (containingthe genomic sequence of the human IgG4 constant region in the pEE12.4vector (Lonza Biologics)). Alternatively, in follow-up constructs,vectors were used containing the fully codon-optimized coding regions ofthe heavy chain (IgG1 or IgG4) in the pEE12.4 vector or the human kappalight chain of HuMab 2F8 or HuMab 7D8 in the pEE6.4 vector (LonzaBiologics).

Example 2 Expression Vectors for the Expression Hinge-Deleted-IgG1-2F8,and Human IgG1 and IgG4 CH2-CH3 Fragments Containing Specific mutations

To introduce mutations in the hinge and CH3 regions of the antibodyheavy chains, Quickchange site-directed mutagenesis kit (Stratagene, LaJolla, Calif.) was used according to the manufacturer's recommendations.Alternatively the constructs were fully synthesized or VH regions werecloned in a vector already containing the specific amino acid encodingsubstitutions.

Constructs encoding the CH2 and CH3 fragments were constructed either byPCR or synthesized fully codon optimized. These constructs had anN-terminal signal peptide and a 6 amino acid His tag and contained aminoacids 341-447 of the human IgG1/4 constant region. The constructs werecloned in pEE12.4.

To construct hinge-deleted-IgG1 (Uni-G1) molecules, a synthetic DNAconstruct were was made encoding the Uni-G1 format for human IgG1isotypes with EGFR specificity. In this construct the natural hingeregion (as defined by the hinge exon) was deleted. An extra Ser to Cysmutation at position 158 was made in the IgG1 construct to salvage theCys bond between the HC and LC chains in this subtype. The proteinsequence is shown below. The construct was inserted in the pEE6.4 vectorand named pHG1-2F8.

QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYGMHWVRQAPGKGLEWVAVIWDDGSYKYYGDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDGITMVRGVMKDYFDYWGQGTLVTVSSASTKGPSVFPLAPCSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Example 3 Expression Vectors for the Expression of Rhesus IgG4-2F8 andIgG4-7D8

Vectors containing the coding regions for the IgG4 heavy and kappa lightchains Chinese Rhesus monkey and the VH and VL regions of Humab 2F8 and7D8 were synthesized, fully codon-optimized and inserted in pEE12.4(heavy chain) and pEE6.4 (light chain). The heavy chain constant regionsequence as used (based on the sequences described by Scinicariello etal., Immunology 111: 66-74, 2004) was the following (aligned to thehuman sequence):

Human IgG4 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHRhesus (Ch) IgG4 -STKGPSVFPLASCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHHuman IgG4 TFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGRhesus (Ch) IgG4 TFPAVLQSSGLYSLSSVVTVPSSSLGTQTYVCNVVHEPSNTKVDKRVEFT--Human IgG4 PPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVRhesus (Ch) IgG4 PPCPACPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVHuman IgG4 QFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVRhesus (Ch) IgG4 QFNWYVDGAEVHHAQTKPRERQFNSTYRVVSVLTVTHQDWLNGKEYTCKVHuman IgG4 SNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYRhesus (Ch) IgG4 SNKGLPAPIEKTISKAKGQPREPQVYILPPPQEELTKNQVSLTCLVTGFYHuman IgG4 PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFRhesus (Ch) IgG4 PSDIAVEWESNGQPENTYKTTPPVLDSDGSYLLYSKLTVNKSRWQPGNIFHuman IgG4 SCSVMHEALHNHYTQKSLSLSLGK Rhesus (Ch) IgG4TCSVMHEALHNHYTQKSLSVSPGKThe Rhesus light chain constant region (CL) sequence used was:

AVAAPSVFIFPPSEDQVKSGTVSVVCLLNNFYPREASVKWKVDGVLKTGNSQESVTEQDSKDNTYSLSSTLTLSSTDYQSHNVYACEVTHQGLSSPVT KSFNRGEC

Example 4 Antibody Production by Transient Expression in HEK-293F Cells

Antibodies were produced, under serum-free conditions, by cotransfectingrelevant heavy and light chain expression vectors in HEK-293F cells(Invitrogen), using 293fectin (Invitrogen), according to themanufacturer's instructions.

Example 5 Purification of IgG1 and IgG4 Antibodies

IgG1 and IgG4 antibodies were purified by protein A affinitychromatography. The cell culture supernatants were filtered over a 0.20μM dead-end filter, followed by loading on a 5 mL Protein A column(rProtein A FF, GE Healthcare, Uppsala, Sweden) and elution of the IgGwith 0.1 M citric acid-NaOH, pH 3. The eluate was immediatelyneutralized with 2 M Tris-HCl, pH 9 and dialyzed overnight to 12.6 mMsodium phosphate, 140 mM NaCl, pH 7.4 (B. Braun, Oss, The Netherlands).After dialysis, samples were sterile filtered over a 0.20 μM dead-endfilter. Concentration of the purified IgGs was determined bynephelometry and absorbance at 280 nm. Purified proteins were analyzedby SDS-PAGE, IEF, mass spectrometry and glycoanalysis.

Example 6 Purification of CH2-CH3 Fragments

The His-tagged CH2-CH3 proteins were purified by immobilized metal ion(Ni²⁺) affinity chromatography (Macherey-Nagel GmbH, Duren, Germany),desalted using PD-10 columns (GE Healthcare) equilibrated with PBS andfiltered-sterilized over 0.2 μM dead-end filters. The concentration ofthe purified proteins was determined by absorbance at 280 nm. Thequality of the purified proteins was analyzed by SDS-PAGE.

Example 7 Generation of Bispecific Antibodies by GSH-Induced Fab-ArmExchange Between Human and Rhesus IgG4 Antibodies

As mentioned above, WO 2008119353 (Genmab) describes an in vitro methodfor producing bispecific antibodies wherein a bispecific antibody isformed by “Fab-arm” or “half-molecule” exchange (swapping of a heavychain and attached light chain) between two monospecific IgG4- orIgG4-like antibodies upon incubation under reducing conditions. ThisFab-arm exchange reaction is the result of a disulfide-bondisomerization reaction wherein the inter heavy-chain disulfide bonds inthe hinge regions of monospecific antibodies are reduced and theresulting free cysteines form a new inter heavy-chain disulfide bondwith cysteine residues of another antibody molecule with a differentspecificity. The resulting product is a bispecific antibody having twoFab arms with different sequences.

To test for Fab-arm exchange between human and rhesus igG4 antibodies,human IgG4-2F8 (anti-EGFR), Human IgG4-7D8 (anti-CD20), Rhesus IgG4-2F8and Rhesus IgG4-7D8 were used to make all possible combinations of twoantibodies. For the in vitro Fab-arm exchange, the antibody mixtures,containing each antibody at a final concentration of 4 μg/mL in 0.5 mLPBS with 0.5 mM reduced glutathione (GSH), were incubated at 37° C. for24 h. To stop the reduction reaction, 0.5 mL PBS/0.05% Tween 20 (PBST)was added to the reaction mixture.

The presence of bispecific antibodies was tested by determination ofbispecific binding using a sandwich enzyme-linked immunosorbent assay(ELISA). ELISA plates (Greiner bio-one, Frickenhausen, Germany) werecoated overnight with 2 μg/mL (100 μL/well) of recombinant extracellulardomain of EGFR in PBS at 4° C. The plates were washed once with PBST.Dilution series of the antibody samples (0-1 μg/mL in 3-fold dilutions)in PBST/0.2% BSA (PBSTB) were transferred to the coated ELISA plates(100 μL/well) and incubated on a plate shaker (300 rpm) for 60 min atroom temperature (RT). Samples were discarded and the plates were washedonce with PBS/0.05% Tween 20 (PBST). Next, the plates were incubated ona plate shaker (300 rpm) with 2 μg/mL mouse anti-idiotypic monoclonalantibody 2F2 SAB1.1 (directed against 7D8; Genmab) in PBTB (100 μL/well)for 60 min. The plates were washed once with PBS/0.05% Tween 20 (PBST).Next, the plates were incubated on a plate shaker (300 rpm) with anHRP-conjugated goat anti-mouse IgG (15G; Jackson ImmunoResearchLaboratories, Westgrove, Pa., USA; 1:5.000) in PBSTB (100 μL/well) for60 min at RT. The plates were washed once with PBS/0.05% Tween 20(PBST). ABTS (50 mg/mL; Roche Diagnostics GmbH, Mannheim, Germany) wasadded (100 μL/well) and incubated protected from light for 30 min at RT.The reaction was stopped with 2% oxalic acid (100 μL/well; Riedel deHaen Seelze, Germany). After 10 min at RT, absorbance at 405 nm wasmeasured in an ELISA plate reader.

FIG. 1 shows that a combination of human and rhesus IgG4 resulted inmore bispecific binding (a higher OD 405 nm) compared with each of thecombinations of IgG4 molecules of the same species. These data show thatFab-arm exchange occurs between human IgG4 and rhesus IgG4. Moreover,the higher bispecific binding suggests that human IgG4 half moleculesshow preferential dimerisation to rhesus IgG4 half molecules(heterodimerization), resulting in an equilibrium of the Fab-armexchange reaction that is shifted towards the bispecific heterodimerinstead of a stochastic exchange with 50% heterodimer and 50%homodimers.

Example 8 Sequence Analysis of Human and Rhesus IgG4

The ability of an antibody to engage in Fab-arm exchange has beendescribed to involve the third constant domain (CH3) in addition to aso-called permissive (for example CPSC containing—) hinge region thatonly requires a reducing environment to be activated (Van der NeutKolfschoten, 2007, Science). For human antibodies, Fab-arm exchange wasfound to be an inherent feature of IgG4, characterized by an arginine(R) at position 409 in the CH3 domain and a permissive hinge(226-CPSC-229) (see WO 2008145142 (Genmab)). In contrast, human IgG1,which does not engage in Fab-arm exchange, has a Lysine (K) at position409 and a stable (i.e. non-permissive) hinge (226-CPPC-229) (EUnumbering, see also FIG. 16).

In an attempt to elucidate the increased Fab-arm exchange between humanand rhesus IgG4 compared to the Fab-arm exchange between IgG4 moleculesof the same species, the core hinge and CH3-CH3 interface amino acids ofhuman and rhesus antibodies were analyzed (see e.g. Dall'Acqua, et al(1998) Biochemistry 37:9266 for an overview of the residues of the humanCH3-CH3 interface). FIG. 2 shows that the core hinge sequence in Chineserhesus IgG4 is 226-CPAC-229 and that the CH3 domain contains a Lysine(K) at position 409. In addition, sequence alignment showed that rhesusIgG4 is characterized by three more amino acid substitutions in theCH3-CH3 interface as compared to human IgG4: isoleucine (I) at position350 in rhesus versus threonine (T) in human; threonine (T) at position370 in rhesus versus lysine (K) in human; and leucine (L) at position405 in rhesus versus phenylalanine (F) in human.

Example 9 Generation of Bispecific Antibodies Using GSH-Induced Fab-ArmExchange Between Human IgG4 and Human IgG1 Containing Rhesus IgG4 CH3Sequences

It has been described for human antibodies that for allowing Fab-armexchange to occur in IgG1 molecules, replacing the IgG1 core hingesequence (CPPC) with the human IgG4 sequence (CPSC) by a P228Ssubstitution had no effect, but that mutating CH3 to an IgG4-likesequence was required for Fab-arm exchange activity (Van der NeutKolfschoten, 2007, Science).

Based on the Fab-arm exchange between human and rhesus IgG4 described inExample 7, it was analyzed whether the Chinese rhesus IgG4 CH3 sequencecould engage human IgG1 for Fab-arm exchange. Therefore, the triplemutation T350I-K370T-F405L (referred to as ITL hereafter) was introducedin human IgG1-2F8 in addition to the P228S mutation that results in thehinge sequence CPSC. The human IgG1-2F8 mutants were combined with humanIgG4-7D8 for in vitro GSH-induced Fab-arm exchange. The antibodymixtures, containing each antibody at a final concentration of 4 μg/mLin 0.5 mL PBS with 0.5 mM GSH, were incubated at 37° C. for 0-3-6-24 h.To stop the reduction reaction, 0.5 mL PBS/0.05 Tween 20 (PBST) wasadded to the reaction mixture. Measurements of bispecific binding in anELISA were performed as described in Example 7.

FIG. 3 confirms that introduction of a CPSC hinge alone does not engagehuman IgG1-2F8 for GSH-induced Fab-arm exchange when combined with humanIgG4-7D8. Also the introduction of the rhesus IgG4-specific CH3interface amino acids (ITL) into human IgG1-2F8, while preserving thewild type IgG1 hinge, did not result in engagement for Fab-arm exchangewhen combined with human IgG4-7D8 under these conditions. In contrast, avariant human IgG1-2F8 backbone sequence that harbors both a CPSCsequence in the hinge and the rhesus IgG4-specific CH3 interface aminoacids (ITL) showed increased bispecific binding after GSH-inducedFab-arm exchange with human IgG4-7D8 compared to two human IgG4antibodies. These data show that a CPSC-containing hinge in combinationwith a CH3 domain containing I, T and L at positions 350, 370 and 405,respectively, is sufficient to engage human IgG1 for GSH-induced Fab-armexchange and that the equilibrium of the exchange reaction is shiftedtowards the exchanged bispecific product when combined with human IgG4.

Example 10 Generation of Bispecific Antibodies by In Vivo Fab-ArmExchange Between Human IgG4 and IgG1 or IgG4 Mutants

To further identify the required characteristics for Fab-arm exchangeengagement, human IgG4 and IgG1 variants were analyzed in vivo. Fourfemale SCID mice (Charles River, Maastricht, The Netherlands) per groupwere i.v. injected with antibody mixtures, containing 600 μg antibody(500 μg 7D8+100 μg 2F8) in a total volume of 300 μL. Blood samples weredrawn from the saphenal vein at 3, 24, 48 and 72 hours after injection.Blood was collected in heparin-containing vials and centrifuged at10,000 g for 5 min to separate plasma from cells. The generation ofbispecific antibodies was followed by assessing CD20 and EGFR bispecificreactivity in an ELISA using serial diluted plasma samples in PBSTB asdescribed in Example 7. Bispecific antibodies in plasma samples werequantified by non-linear regression curve-fitting (GraphPad Software,San Diego, Calif.) using an in vitro exchanged antibody mixture asreference.

FIG. 4 shows that human IgG4-2F8, in which either the hinge or the CH3sequence is converted to the corresponding human IgG1 sequence (CPPC orR409K, respectively), does not engage in Fab-arm exchange anymore invivo. Vice versa, human IgG1, in which both the hinge region and the CH3interface sequences are converted to the corresponding human IgG4sequences (CPSC and K409R), is able to participate in Fab-arm exchangein vivo. These data show that a CPSC-containing hinge (S at position228) in combination with a CH3 domain containing an arginine (R) atposition 409 is enough to enable Fab-arm exchange by human IgG1 in vivo.

Example 11 Generation of Bispecific Antibodies by 2-MEA-Induced Fab-ArmExchange: Bypass/Disruption of a Stabilized Hinge

2-Mercaptoethylamine.HCl (2-MEA) is a mild reducing agent that has beendescribed to selectively cleave disulphide bonds in the hinge region ofantibodies, while preserving the disulphide bonds between the heavy andlight chains. Therefore, a concentration series of 2-MEA was tested forits ability to induce the generation of bispecific antibodies by Fab-armexchange between two antibodies containing CPSC or CPPC hinge regions.The antibody mixtures, containing each antibody at a final concentrationof 0.5 mg/mL, were incubated with a concentration series of 2-MEA (0,0.5, 1.0, 2.0, 5.0, 7.0, 10.0, 15.0, 25.0 and 40.0 mM) in a total volumeof 100 μL TE at 37° C. for 90 min. To stop the reduction reaction, thereducing agent 2-MEA was removed by desalting the samples using spincolumns (Microcon centrifugal filters, 30 k, Millipore) according to themanufacturer's recommendations. Bispecific binding was measured in anELISA as described in Example 7.

2-MEA-induced Fab-arm exchange was tested for the combinationIgG4-2F8×IgG4-7D8, containing CPSC hinge regions and known toparticipate in GSH-induced Fab-arm exchange, and for the combinationIgG1-2F8-ITL×IgG4-7D8-CPPC, not participating in GSH-induced Fab-armexchange due to the stabilized hinge regions (described in Example 9,FIG. 3). Surprisingly, 2-MEA was found to induce separation of lightchains from heavy chains as determined by non-reducing SDS-PAGE (datanot shown). Nonetheless, functional bispecific antibodies were generatedas shown in FIG. 5. The maximal level of bispecific binding afterFab-arm exchange between wild type human IgG4-2F8 and IgG4-7D8 wasreached at a concentration of 2.0 mM 2-MEA and was comparable to thelevel reached with 0.5 mM GSH as described in Example 9 (FIG. 3).However, 2-MEA was able to induce Fab-arm exchange between the humanantibodies IgG1-2F8-ITL and IgG4-7D8-CPPC (with stabilized hingeregions) in a dose-dependent manner. While little or no bispecificantibodies were formed at low 2-MEA concentrations, probably due to thepresence of a CPPC sequence in the hinge region of both antibodies, thegeneration of bispecific antibodies was very efficient at higherconcentrations of 2-MEA. Maximal bispecific binding was reached at 25 mM2-MEA and exceeded maximal binding after Fab-arm exchange between thetwo wild type IgG4 antibodies. These maximal binding levels werecomparable to what is described in Example 9 (FIG. 3) for GSH treatmentof the corresponding antibody with a CPSC hinge (IgG1-2F8-CPSC-ITL). AsIgG1-2F8-ITL and IgG4-7D8-CPPC both contain a CPPC hinge, these dataindicate that 2-MEA could bypass the requirement of a CPSC hinge for invitro Fab-arm exchange.

Example 12 Mass Spectrometry to Follow the Generation of BispecificAntibodies by 2-MEA-Induced Fab-Arm Exchange

The generation of bispecific antibodies by 2-MEA-induced Fab-armexchange is described in Example 11, where bispecific binding was shownby an ELISA (FIG. 5). To confirm that bispecific antibodies are formed,the samples were analyzed by electrospray ionization mass spectrometry(ESI-MS) to determine the molecular weights. First, samples weredeglycosylated by incubating 200 μg antibody overnight at 37° C. with0.005 U N-Glycanase (cat.no. GKE-5006D; Prozyme) in 180 μL PBS. Sampleswere desalted on an Aquity UPLC™ (Waters, Milford, USA) with a BEH300C18, 1.7 μm, 2.1×50 mm column at 60° C. and eluted with a gradient of amixture of MQ water (Eluens A) and LC-MS grade acetonitrile (eluens B)(Biosolve, Valkenswaard, The Netherlands) containing 0.05% formic acid(Fluka Riedel-de Haën, Buchs, Germany). Time-of-flight electrosprayionization mass spectra were recorded on-line on a micrOTOF™ massspectrometer (Bruker, Bremen, Germany) operating in the positive ionmode. Prior to analysis, a 500-4000 m/z scale was calibrated with EStuning mix (Agilent Technologies, Santa Clara, USA). Mass spectra weredeconvoluted by using Maximal Entropy that is provided with theDataAnalysis™ software v. 3.4 (Bruker, Bremen, Germany). Based on themolecular mass of the antibodies used for Fab-arm exchange in thisexperiment, the bispecific antibodies could be discriminated from theoriginal antibodies (also described in Example 15, FIG. 9C forIgG1-2F8-ITL×IgG4-7D8-CPPC). For the peak of bispecific antibody, thearea under the curve was determined and divided by the total area underthe curves to calculate the percentage bispecific antibody in eachsample. FIG. 6A shows three representative mass spectrometry profiles ofthe Fab-arm exchange reaction between IgG1-2F8-ITL and IgG4-7D8-CPPCwith 0 mM 2-MEA (two peaks corresponding to the parental antibodies), 7mM 2-MEA (three peaks corresponding to the parental and the bispecificantibodies), and 40 mM 2-MEA (one peak corresponding to the bispecificantibody). The homogenous peak of the bispecific product indicates thatno light chain mispairing occurred, which would have resulted insubdivided peaks. The quantified data are presented in FIG. 6B and showthat Fab-arm exchange between IgG1-2F8-ITL and IgG4-7D8-CPPC resulted innearly 100% bispecific antibody. In contrast, Fab-arm exchange betweenwild type IgG4 antibodies resulted in less than 50% bispecific product.These data confirm the results from the bispecific binding ELISAdescribed in Example 11 (FIG. 5).

Example 13 Stability of Bispecific Antibodies Generated by 2-MEA-InducedFab-Arm Exchange

The stability of bispecific antibodies generated by 2-MEA-induced invitro Fab-arm exchange was tested. Therefore, 2 μg of a bispecificsample generated from IgG1-2F8-ITL and IgG4-7D8-CPPC with 7.0 mM 2-MEA(as described in Example 11, FIG. 5) was used in a GSH-induced Fab-armexchange reaction in the presence of a concentration series (0, 2, 20,100 μg) irrelevant IgG4 (IgG4-MG against acetylcholine receptor),representing a 0, 1, 10, 50× excess of IgG4-MG compared to the 2 μgbispecific test sample. Fab-arm exchange in this reaction would resultin loss of bispecific EGFR/CD20 binding. The conditions for the GSHreduction reaction were the same as described in Example 7 (24 h at 37°C. in 0.5 mL PBS/0.5 mM GSH). To stop the reduction reaction, 0.5 mLPBSTB was added to the reaction mixture. Bispecific binding was measuredin an ELISA as described in Example 7. Bispecific binding after the GSHreduction reaction is presented relative to the bispecific bindingmeasured in the starting material (control), which was set to 100%.

FIG. 7A shows that for the IgG1-2F8-ITL×IgG4-7D8-CPPC derived bispecificsample, EGFR/CD20 bispecific binding is not significantly changed afterGSH-induced Fab-arm exchange in the presence of irrelevant IgG4. Thisindicates that the bispecific product is stable, i.e. does notparticipate in GSH-induced Fab-arm exchange. As a control, FIG. 7B showsthat an IgG4-2F8×IgG4-7D8 derived sample shows diminished EGFR/CD20bispecific binding after GSH-induced Fab-arm exchange in the presence ofirrelevant IgG4, indicating that this product is not stable. These datashow that the heterodimer consisting of a human IgG1 heavy chaincontaining the triple mutation T350I-K370T-F405L in the CH3 domain, anda human IgG4 heavy chain containing the S228P substitution resulting ina stabilized hinge (CPPC), is stable.

Example 14 In Vivo Analysis of the Pharmacokinetics and Stability ofBispecific Antibodies Generated by 2-MEA-Induced Fab-Arm Exchange

The bispecific antibody generated by in vitro 2-MEA-induced Fab-armexchange between IgG1-2F8-ITL×IgG4-7D8-CPPC was injected in SCID mice toanalyze its stability (in vivo Fab-arm exchange) and pharmacokineticproperties (plasma clearance rate) in comparison to the parentalantibodies IgG1-2F8-ITL and IgG4-7D8-CPPC. Three groups of mice (3 miceper group) were injected intravenously in the tail vein with 200 μLpurified antibody: (1) 100 μg bispecific antibody; (2) 100 μg bispecificantibody+1,000 μg irrelevant IgG4 (natalizumab, anti-α4-integrin); (3)50 μg IgG1-2F8-ITL+50 μg IgG4-7D8-CPPC. Blood samples (50-100 μL) werecollected by cheek puncture at pre-determined time intervals afterantibody administration (10 min, 3 h, 1, 2, 7, 14, 21 days). Blood wascollected into heparin containing vials and centrifuged for 10 min at14,000 g. Plasma was stored at −20° C. before further analysis.

Total IgG concentrations in the plasma samples were assayed by ELISA.The assay conditions of the succeeding steps were the same as for theELISA described in Example 7. Specific compounds used for total IgGmeasurement were the following: coat with 2 μg/mL mouse anti-human IgG(clone MH16-1; CLB; cat. no. M1268); serum samples dilutions (1:500 and1:2,500 for groups 1 and 3) and (1:2,500 and 1:10,000 for group 2);conjugate:HRP-conjugated goat anti-human IgG (clone 11H; Jackson; cat.no. 109-035-098; 1:10,000). The presence of bispecific antibodies in theplasma samples was assayed and quantified by CD20 and EGFR bispecificreactivity in an ELISA as described in Example 10.

FIG. 8A shows total antibody plasma concentrations. The shape of theplasma clearance curves was identical in all groups, indicating that theplasma clearance of the bispecific antibody was the same as for theparental antibodies IgG1-2F8-ITL and IgG4-7D8-CPPC over the analyzedtime interval. FIG. 8B shows the plasma concentrations of bispecificantibodies over time. The addition of a 10-fold excess irrelevant IgG4to the bispecific antibody did not affect bispecific antibodyconcentrations, indicating that no Fab-arm exchange occurred in vivo.After injection of the parental antibodies (IgG1-2F8-ITL+IgG4-7D8-CPPC),no bispecific antibodies were detectable in the plasma, confirming thatthese antibodies do not participate in Fab-arm exchange in vivo. Thesedata indicate that the bispecific antibody product, generated by invitro 2-MEA-induced Fab-arm exchange between IgG1-2F8-ITL×IgG4-7D8-CPPC,was stable in vivo (no Fab-arm exchange) and showed comparablepharmacokinetic properties (plasma clearance rate) as the parentalmonovalent antibodies.

Example 15 Purity of the Bispecific Antibody Generated by 2-MEA-InducedFab-Arm Exchange Between Two Antibodies

A batch of bispecific antibody, generated by 2-MEA-induced Fab-armexchange between human IgG1-2F8-ITL×IgG4-7D8-CPPC, was purified on aPD-10 desalting column (cat.no. 17-0851-01; GE Healthcare). Next, thepurity of the bispecific product was analyzed by sodium dodecyl sulfatepolyacrylamide gelelectrophoresis (SDS-PAGE), high performance sizeexclusion chromatography (HP-SEC) and mass spectrometry. Thefunctionality of the generated bispecific antibody was confirmed bybispecific binding in an ELISA (data not shown).

SDS-PAGE was performed under reducing and non-reducing conditions on4-12% NuPAGE Bis-Tris gels (Invitrogen, Breda, The Netherlands) using amodified Laemli method (Laemli 1970 Nature 227(5259): 680-5), where thesamples were run at neutral pH. The SDS-PAGE gels were stained withCoomassie and digitally imaged using the GeneGenius (Synoptics,Cambridge, UK). FIG. 9A shows that the antibody sample after Fab-armexchange consists of intact IgG, with a trace of half molecules (H1L1)detectable on the non-reduced gel (FIG. 9A-b).

HP-SEC fractionation was performed using a Waters Alliance 2695separation unit (Waters, Etten-Leur, The Netherlands) connected to a TSKHP-SEC column (G3000SW_(xl); Toso Biosciences, via Omnilabo, Breda, TheNetherlands) and a Waters 2487 dual λ absorbance detector (Waters). Thesamples were run at 1 mL/min. Results were processed using Empowersoftware version 2002 and expressed per peak as percentage of total peakheight. FIG. 9B shows that >98% of the sample consists of intact IgG,with practically no aggregates formed.

Mass spectrometry was performed as described in Example 12. FIG. 9Cshows the mass spectrometry profiles of the starting materialsIgG1-2F8-ITL and IgG4-7D8-CPPC and the bispecific product generated byFab-arm exchange between IgG1-2F8-ITL×IgG4-7D8-CPPC. The product in theFab-arm exchanged sample is 145,901 kDa, which perfectly matches withthe bispecific product derived from IgG1-2F8-ITL(146,259.5/2=73,130)+IgG4-7D8-CPPC (145,542.0/2=72,771). Moreover, thebispecific antibody product showed a homogenous peak, indicating that nolight chain mispairing occurred, which would have resulted in subdividedpeaks. These data show that Fab-arm exchange resulted in 100% bispecificantibody. The small peaks detected in addition to the main peak (K0) ofthe IgG4-7D8-CPPC and bispecific sample can be attributed to thepresence of one (K1) or two (K2) C-terminal lysines.

These data show that a ˜100% functional bispecific antibody sample wasgenerated by 2-MEA-induced Fab-arm exchange betweenIgG1-2F8-ITL×IgG4-7D8-CPPC.

Example 16 Unraveling the Requirement of the T350I, K370T and F405LSubstitutions for Fab-Arm Exchange Engagement of Human IgG1

To further identify the determinants in the IgG1 CH3 domain that arerequired for IgG1 to be engaged in Fab-arm exchange, IgG1 containing thetriple mutation T350I-K370T-F405L (ITL) was compared to the doublemutants T350I-K370T (IT), T350I-F405L (IL) and K370T-F405L (TL). Alsothe single mutant F405L (L) was tested. 2-MEA was used as a reductant toinduce in vitro Fab-arm exchange (50 μg of each antibody in 100 μLPBS/25 mM 2-MEA for 90 min at 37° C.). For the single mutant F405Lantibody, unpurified antibody from supernatant of a transienttransfection was used after buffer-exchange to PBS using Amicon Ultracentrifugal devices (30 k, Millipore, cat. no. UFC803096). To stop thereduction reaction, the reducing agent 2-MEA was removed by desaltingthe samples using spin columns as described in Example 11. Thegeneration of bispecific antibodies was determined by bispecific bindingmeasured in an ELISA as described in Example 7.

The triple (ITL), double mutations (IT, IL and TL) and single mutation(L) were introduced in IgG1-2F8. These mutants were combined withIgG4-7D8, containing a CPSC hinge (wild type) or a stabilized hinge(IgG4-7D8-CPPC), for Fab-arm exchange using 25 mM 2-MEA for 90 min at37° C. FIG. 10A-B shows that the IgG1-2F8-IL and -TL mutants showedFab-arm exchange to the same level as the triple mutant ITL,irrespective of the combined IgG4-7D8 (CPSC or CPPC hinge). In contrast,no bispecific binding was found for the combination with the IgG1-2F8-ITmutant. FIG. 10C shows that also the IgG1-2F8-F405L mutant showedFab-arm exchange, irrespective of the combined IgG4-7D8 (CPSC or CPPChinge). These data indicate that the F405L mutation is sufficient toengage human IgG1 for Fab-arm exchange under the conditions mentionedabove.

Example 17 Generation of Bispecific Antibodies by 2-MEA-Induced Fab-ArmExchange at Different Temperatures

The ability of 2-MEA to induce the generation of bispecific antibodiesby Fab-arm exchange between two different antibodies, was tested atdifferent temperatures. The Fab-arm exchange reactions were started byincubating 160 μg human IgG1-2F8-ITL with 160 μg IgG4-7D8-CPPC in 320 μlPBS/25 mM 2-MEA (final concentration of 0.5 mg/mL for each antibody) ateither 0° C., 20° C. (RT) or 37° C. From these reactions, 20 μL sampleswere taken at different time points (0, 2.5, 5, 10, 15, 30, 45, 60, 75,90, 120, 150, 180 and 240 min). 20 μL PBS was added to each samplebefore the reducing agent 2-MEA was removed by desalting the samplesusing a Zeba 96-well spin desalting plate (7 k, cat#89808 Thermo FisherScientific), according to the manufacturer's recommendations. The totalantibody concentrations were determined by measuring absorbance at 280nm wavelength using a Nanodrop ND-1000 spectrophotometer (Isogen LifeScience, Maarssen, The Netherlands). Dilution series of the antibodysamples (total antibody concentration 0-20 μg/mL in 25-fold dilutions)were used in an ELISA to measure bispecific binding as described inExample 7.

FIG. 11 shows that the generation of bispecific antibodies by2-MEA-induced Fab-arm exchange between human IgG1-2F8-ITL andIgG4-7D8-CPPC was found to be most efficient at 37° C., with maximalbispecific binding reached after 45 min. At room temperature, thegeneration of bispecific antibodies was slower, reaching maximalbispecific binding after 240 min. At 0° C., no generation of bispecificbinding was observed during the analyzed time course.

Example 18 Analysis of Different Reducing Agents for their Ability toInduce the Generation of Bispecific Antibodies by In Vitro Fab-ArmExchange

It has been shown above that 0.5 mM GSH can induce in vitro Fab-armexchange between human IgG4 and IgG1-CPSC-ITL, but not between humanIgG4 and IgG1-ITL containing a stable hinge (FIG. 3). In addition, 2-MEAwas found to be able to induce Fab-arm exchange between antibodies withstabilized hinge regions, such as IgG1-ITL×IgG4-CPPC (FIG. 5). To testwhether other concentrations of GSH or 2-MEA or other reducing agentsare capable of inducing in vitro Fab-arm exchange between two differentantibodies, concentration series of 2-MEA, GSH and DTT (dithiothreitol)were tested. Therefore, combinations of 10 μg human IgG1-2F8-ITL and 10μg IgG4-7D8-CPPC in 20 μl PBS (final concentration of 0.5 mg/mL for eachantibody) were incubated at 37° C. with concentration series of thedifferent reducing agents (0.0, 0.04, 0.1, 0.2, 0.5, 1.0, 2.5, 5.0,12.5, 25.0 and 50.0 mM). After 90 min, 20 μL PBS was added to eachsample and the reducing agent was removed by desalting the samples usingspin desalting plate as described in Example 17. Total antibodyconcentrations were determined as described in Example 17. Dilutionseries of the antibody samples (total antibody concentration 0-20 μg/mLin 3-fold dilutions) were used in an ELISA to measure bispecific bindingas described in Example 7.

FIG. 12 confirms that 2-MEA induces maximal bispecific binding at aconcentration of 25 mM 2-MEA. DTT was found to be very effective in thegeneration of bispecific antibodies with maximal bispecific bindingreached at 2.5 mM DTT. GSH concentrations in the range 0-5 mM were notable to induce the generation of bispecific antibodies by Fab-armexchange between the IgG1-ITL and IgG4-CPPC antibodies, both containingstabilized hinge regions. Higher GSH concentrations (12.5-50 mM)resulted in the formation of antibody aggregates, as was determined bynon-reducing SDS-PAGE (data not shown). Therefore, these samples wereexcluded from the analysis. These data show that the generation ofbispecific antibodies by Fab-arm exchange between two differentantibodies can be induced by different reducing agents.

Example 19 Determinants at the IgG1 409 Position for Engagement in2-MEA-Induced Fab-Arm Exchange in Combination with IgG1-ITL

2-MEA can induce Fab-arm exchange between human IgG1-ITL and IgG4-CPPC,as described in Example 11 (FIG. 5). The CH3 interface residues of humanIgG1 and IgG4 differ at position 409 only: lysine (K) in IgG1 andarginine (R) in IgG4 (described in Example 8, FIG. 2). Therefore, it wastested whether substitution of lysine at position 409 by arginine or anyother amino acid (K409X) could enable IgG1 to engage in 2-MEA-inducedFab-arm exchange with IgG1-ITL. Combinations of 10 μg human IgG1-2F8-ITLand 10 μg IgG1-7D8-K409X in 20 μl PBS/25 mM 2-MEA (final concentrationof 0.5 mg/mL for each antibody) were incubated for 90 min at 37° C.Unpurified antibodies from supernatants of transient transfections wereused after buffer-exchange to PBS using Amicon Ultra centrifugal devices(30 k, Millipore, cat. no. UFC803096). After the Fab-arm exchangereaction, 20 μL PBS was added to each sample and the reducing agent wasremoved by desalting the samples using spin desalting plate as describedin Example 17. Dilution series of the antibody samples (total antibodyconcentration 0-20 μg/mL in 3-fold dilutions) were used in an ELISA tomeasure bispecific binding as described in Example 7.

FIG. 13A shows the results of bispecific binding upon 2-MEA inducedFab-arm exchange between IgG1-2F8-ITL×IgG1-7D8-K409X. In FIG. 13B, theexchange is presented as bispecific binding relative to a purified batchof bispecific antibody derived from a 2-MEA-induced Fab-arm-exchangebetween IgG1-2F8-ITL and IgG4-7D8-CPPC, which was set to 100%. Thesedata were also scored as (−) no Fab-arm exchange, (+/−) low, (+)intermediate or (++) high Fab-arm exchange, as presented in Table 1. NoFab-arm exchange (−) was found when the 409 position in IgG1-7D8 was K(=wild type IgG1), L or M. Fab-arm exchange was found to be intermediate(+) when the 409 position in IgG1-7D8 was F, I, N or Y and high (++)when the 409 position in IgG1-7D8 was A, D, E, G, H, Q, R, S, T, V or W.

TABLE 1 2-MEA-induced Fab-arm exchange between IgG1-2F8-ITL andIgG1-7D8-K409X mutants. Fab-arm exchange IgG1-7D8-K409X x IgG1-2F8-ITL A++ D ++ E ++ F + G ++ H ++ I + K − L − M − N + Q ++ R ++ S ++ T ++ V ++W ++ Y + The generation of bispecific antibodies after 2-MEA-induced invitro Fab-arm exchange between IgG1-2F8-ITL and IgG1-7D8-K409X mutantswas determined by a sandwich ELISA. (−) no, (+/−) low, (+) intermediate,(++) high Fab-arm exchange.

Example 20 Antibody Deglycosylation does not Influence the Generation ofBispecific Antibodies by 2-MEA-Induced Fab-Arm Exchange

IgG4-7D8 and IgG4-7D8-CPPC samples were deglycosylated by incubating 200μg antibody overnight at 37° C. with 0.005 U N-Glycanase (cat.no.GKE-5006D; Prozyme) in 180 μL PBS. These samples were used directly in aFab-arm exchange reaction. Fab-arm exchange was performed by incubating50 μg of each antibody in 100 μl PBS/25 mM 2-MEA (final concentration of0.5 mg/mL for each antibody) for 90 min at 37° C. The reducing agent2-MEA was removed by desalting the samples using spin columns asdescribed in Example 11. Dilution series of the antibody samples (totalantibody concentration 0-20 μg/mL in 3-fold dilutions) were used in asandwich ELISA to measure bispecific binding as described in Example 7.

Mass spectrometry analysis showed that the deglycosylation reactionresulted in 100% deglycosylated antibody product (data not shown). FIG.14 shows that Fab-arm exchange involving deglycosylated antibodies didnot differ from Fab-arm exchange with the corresponding glycosylatedantibodies (IgG4-2F8×IgG4-7D8-deglycosylated versus IgG4-2F8×IgG4-7D8and IgG1-2F8-ITL×IgG4-7D8-CPPC-deglycosylated versusIgG1-2F8-ITL×IgG4-7D8-CPPC). These data indicate that deglycosylationdid not affect the generation of bispecific antibodies by 2-MEA-inducedFab-arm exchange.

Example 21 Quantification of the Non-Covalent CH3-CH3 Interaction

The strength of the interactions at the CH3 interface should be suchthat it is possible that both heavy chains in the parental antibodiesdissociate in the Fab-arm exchange reaction and that they subsequentlyassociate in the heterodimerization reaction. Therefore, the correlationbetween the ability to participate in Fab-arm exchange and the strengthof the non-covalent CH3-CH3 interaction (dissociation constant, K_(D))was analyzed. GSH-induced Fab-arm exchange was performed as described inExample 9 (0.5 mM GSH at 37° C.) for the following combinations of humanantibodies:

IgG1-2F8×IgG1-7D8

IgG1-2F8-CPSC×IgG1-7D8-CPSC

IgG1-2F8-CPSC-T350I×IgG1-CPSC-7D8-T350I

IgG1-2F8-CPSC-K370T×IgG1-7D8-CPSC-K370T

IgG1-2F8-CPSC-ITL×IgG1-7D8-CPSC-ITL

IgG1-2F8-CPSC-K409R×IgG1-7D8-CPSC-K409R

IgG4-2F8×IgG4-7D8

IgG4-2F8-R409K×IgG4-7D8-R409K

IgG4-2F8-R409A×IgG4-7D8-R409A

IgG4-2F8-R409L×IgG4-7D8-R409L

IgG4-2F8-R409M×IgG4-7D8-R409M

IgG4-2F8-R409T×IgG4-7D8-R409T

IgG4-2F8-R409W×IgG4-7D8-R409W

IgG4-2F8-F405A×IgG4-7D8-F405A

IgG4-2F8-F405L×IgG4-7D8-F405L

IgG4-2F8-Y349D×IgG4-7D8-Y349D

IgG4-2F8-L351K×IgG4-7D8-L351K

IgG4-2F8-E357T×IgG4-7D8-E357T

IgG4-2F8-S364D×IgG4-7D8-S364D

IgG4-2F8-K370Q×IgG4-7D8-K370Q

IgG4-2F8-K370E×IgG4-7D8-K370E

The generation of bispecific antibodies was measured by determination ofbispecific binding in a sandwich ELISA as described in Example 7. FIGS.15A/B/C show the results of the bispecific binding after the Fab-armexchange reaction.

To measure the effect of the above mentioned CH3 mutations on thestrength of the CH3-CH3 interaction, fragments composed of only theCH2-CH3 domains were made. The lack of a hinge region in these fragmentsprevented covalent inter-heavy chain disulfide bonds. The fragments wereanalyzed by native mass spectrometry. Samples were buffer-exchanged to100 mM ammonium acetate pH 7, using 10 kDa MWCO spin-filter columns.Aliquots (˜1 μL) of serial diluted samples (20 μM-25 nM; monomerequivalent) were loaded into gold-plated borosilicate capillaries foranalysis on a LCT mass spectrometer (Waters). The monomer signal, M_(s),was defined as the area of the monomer peaks as a fraction of the areaof all peaks in the spectrum (M_(s)/(M_(s)+D_(s)) where D_(s)=the dimersignal). The concentration of monomer at equilibrium, [M]_(eq), wasdefined as M_(s)·[M]₀ where [M]₀ is the overall protein concentration interms of monomer. The dimer concentration at equilibrium, [D]_(eq), wasdefined as ([M]₀−[M]_(eq))/2. The K_(D), was then extracted from thegradient of a plot of [D]_(eq) versus [M]_(eq) ². The K_(D) of thenon-covalent CH3-CH3 interactions is presented in Table 2.

The correlation between the ability to engage in Fab-arm exchange andthe strength of the non-covalent CH3-CH3 interactions was analyzed.FIGS. 15D/E show the percentage bispecific binding after Fab-armexchange plotted against the measured K_(D) of the corresponding CH2-CH3fragment (FIG. 15D for IgG1; FIG. 15E for IgG4). These data suggest thatunder the tested conditions there is a specific range of apparent K_(D)values of the CH3-CH3 interaction that allows efficient Fab-armexchange.

TABLE 2 The K_(D) of the non-covalent CH3—CH3 interactions CH2—CH3construct K_(D) (M) fold-difference* G1 3.0 × 10⁻⁹ 1.0000 G1-T350I 7.0 ×10⁻⁹ 0.4000 G1-K370T 4.5 × 10⁻⁸ 0.0700 G1-ITL 1.0 × 10⁻⁶ 0.0030 G1-K409R1.1 × 10⁻⁷ 0.0300 G4 4.8 × 10⁻⁸ 1.0000 G4-R409K 8.0 × 10⁻⁹ 6.0000G4-R409A 1.6 × 10⁻⁷ 0.3000 G4-R409L 1.5 × 10⁻⁸ 3.2000 G4-R409M 3.0 ×10⁻⁹ 16.0000 G4-R409T 7.2 × 10⁻⁷ 0.0700 G4-R409W 3.4 × 10⁻⁵ 0.0014G4-F405A 1.9 × 10⁻⁵ 0.0025 G4-F405L 2.5 × 10⁻⁵ 0.0019 G4-L351K 7.4 ×10⁻⁷ 0.0600 G4-E357T 4.1 × 10⁻⁵ 0.0012 G4-S364D 4.7 × 10⁻⁸ 1.0200G4-K370Q 1.1 × 10⁻⁸ 4.3000 G4-K370E 2.0 × 10⁻⁹ 24.0000 *Compared to thecorresponding CH2—CH3 fragments of wild type IgG1 or IgG4

Example 22 Analysis of Different Reductantia for their Ability to Inducethe Generation of Bispecific Antibodies by In Vitro Fab-Arm-ExchangeBetween IgG1-2F8-F405L and IgG1-7D8-K409R

2-MEA and DTT were found to induce in vitro Fab-arm-exchange betweenhuman IgG1-ITL and IgG4-CPPC (FIG. 12). It was tested whether thesereductantia can also induce in vitro Fab-arm-exchange between humanIgG1-2F8-F405L and IgG1-7D8-K409R. Concentration series of 2-MEA, DTT,GSH and TCEP (tris(2-carboxyethyl)phosphine) were tested.Fab-arm-exchange was performed as described in Example 18. The testedconcentration series of the different reducing agents were as follows:0.0, 0.04, 0.1, 0.2, 0.5, 1.0, 5.0, 25.0, 50.0 mM 2-MEA, GSH, DTT orTCEP.

FIG. 17 confirms that 2-MEA induces maximal Fab-arm-exchange at aconcentration of 25 mM 2-MEA, which persisted at the higherconcentration of 50.0 mM 2-MEA. DTT was found to be very effective inthe generation of bispecific antibodies with maximal Fab-arm-exchangereached at 0.5 mM DDT, which also persisted over higher concentrationsof DTT (1.0-50.0 mM). Also TCEP was found to be very effective in thegeneration of bispecific antibodies with maximal Fab-arm-exchangereached at 0.5 mM. At a concentration ≧25.0 mM, Fab-arm-exchange by TCEPwas disturbed. GSH concentrations in the range 0.0-5.0 mM were not ableto induce the generation of bispecific antibodies by Fab-arm-exchange.Higher GSH concentrations (25.0-50.0 mM) resulted in the formation ofantibody aggregates (data not shown). Therefore, these samples wereexcluded from the analysis. These data show that the generation ofbispecific antibodies by Fab-arm-exchange between two differentantibodies can be induced by different reducing agents.

Example 23 Generation of Bispecific Antibodies by 2-MEA-InducedFab-Arm-Exchange Between IgG1-2F8-F405L and IgG1-7D8-K409R

To confirm the formation of bispecific antibodies by 2-MEA-inducedFab-arm exchange between human IgG1-2F8-F405L and IgG1-7D8-K409R, themolecular weights of samples from the Fab-arm-exchange reactions with aconcentration series of 2-MEA were determined by ESI-MS. The testedconcentration series was as follows: 0.0, 0.5, 1.0, 2.0, 5.0, 7.0, 10.0,15.0, 25.0 and 40.0 mM 2-MEA. Fab-arm-exchange (in PBS) and sandwichELISA were performed as described in Example 11. ESI-MS was performed asdescribed in Example 12.

FIG. 18A shows that 2-MEA induced Fab-arm-exchange betweenIgG1-2F8-F405L and IgG1-7D8-K409R in a dose-dependent manner,efficiently leading to the generation of bispecific antibodies with amaximal level of bispecific binding at a concentration of 15.0 mM 2-MEA.The quantified ESI-MS data are presented in FIG. 18B and show thatFab-arm-exchange between IgG1-2F8-F405L and IgG1-7D8-K409R resulted innearly 100% bispecific antibody, confirming the results from thebispecific-binding ELISA.

Example 24 Purity of the Bispecific Antibody Generated by 2-MEA-InducedFab-Arm-Exchange Between Human IgG1-2F8-F405L×IgG1-7D8-K409R

A batch of bispecific antibody, generated by 2-MEA-inducedFab-arm-exchange between human IgG1-2F8-F405L×IgG1-7D8-K409R, waspurified using a PD-10 desalting column (cat.no. 17-0851-01; GEHealthcare). Next, the purity of the bispecific product was analyzed bymass spectrometry as described in Example 12.

FIG. 19 shows the mass spectrometry profiles of the starting materialsIgG1-2F8-F405L and IgG1-7D8-K409R and the bispecific product generatedby Fab-arm-exchange between IgG1-2F8-F405L×IgG1-7D8-K409R. The productin the Fab-arm-exchanged sample is 146,160.7 kDa, which matches with thebispecific product derived from IgG1-2F8-F405L(146,606.8/2=73,303.3)×IgG1-7D8-K409R (146,312.2/2=73,156.1)=146,459.4kDa. Moreover, the bispecific antibody product showed a homogenous peak,indicating that no light chain mispairing occurred, which would haveresulted in subdivided peaks. These data show that Fab-arm-exchangeresulted in approximately 100% bispecific antibody.

Example 25 In Vivo Analysis of the Stability and Pharmacokinetics ofBispecific Antibodies Generated from IgG1-2F8-F405L×IgG1-7D8-K409R by2-MEA-Induced Fab-Arm-Exchange

The bispecific antibody generated by in vitro 2-MEA-inducedFab-arm-exchange between IgG1-2F8-F405L×IgG1-7D8-K409R was injected inSCID mice to analyze its stability (in vivo Fab-arm-exchange) andpharmacokinetic properties as described in Example 14. Two groups ofmice (3 mice per group) were analyzed: (1) 100 μg bispecific antibody;(2) 100 μg bispecific antibody+1,000 μg irrelevant IgG4 (IgG4-637,described in WO2007068255). Total IgG concentrations in the plasmasamples were assayed by ELISA as described in Example 14, with theexception that in this example, HRP-conjugated goat anti-human IgG(Jackson, cat. no. 109-035-098, 1/10,000) was used as a conjugate fordetection. The presence of bispecific antibodies in the plasma sampleswas assayed and quantified by CD20 and EGFR bispecific reactivity in asandwich ELISA as described in Example 14.

FIG. 20A shows total antibody plasma concentrations over time. The shapeof the plasma clearance curves was identical in both groups. FIG. 20Bshows the plasma concentrations of bispecific antibody over time. Theaddition of a 10-fold excess irrelevant IgG4 to the bispecific antibodydid not affect bispecific antibody concentrations, indicating that noFab-arm-exchange occurred in vivo. These data indicate that thebispecific antibody product, generated by in vitro 2-MEA-inducedFab-arm-exchange between IgG1-2F8-F405L×IgG1-7D8-K409R, was stable invivo (no Fab-arm-exchange).

Example 26 CDC-Mediated Cell Kill by Bispecific Antibody Generated by2-MEA-Induced Fab-Arm-Exchange Between HumanIgG1-2F8-F405L×IgG1-7D8-K409R

The CD20 antibody IgG1-7D8 can efficiently kill CD20-expressing cells bycomplement-dependent cytotoxicity (CDC). In contrast, the EGFR antibodyIgG1-2F8 does not mediate CDC on target cells expressing EGFR. It wastested whether the mutant IgG1-7D8-K409R and the bispecific antibodygenerated by 2-MEA-induced Fab-arm-exchange betweenIgG1-2F8-F405L×IgG1-7D8-K409R were still able to induce CDC onCD20-expressing cells. 10⁵ Daudi or Raji cells were pre-incubated for 15min with a concentration series of antibody in 80 μL RPMI mediumsupplemented with 0.1% BSA in a shaker at room temperature. 20 μL normalhuman serum (NHS) was added as a source of complement (20% NHS finalconcentration) and incubated for 45 min at 37° C. 30 μL ice cold RPMImedium supplemented with 0.1% BSA was added to stop the CDC reaction.Dead and viable cells were discriminated by adding 10 μL 10 μg/mLpropidium iodide (PI) (1 μg/mL final concentration) and FACS analysis.

FIG. 21 shows that CDC-mediated cell kill of CD20-expressing Daudi (FIG.21A) and Raji (FIG. 21B) cells by IgG1-7D8 was not influenced by theintroduction of the K409R mutation. Both Daudi and Raji cells do notexpress EGFR, resulting in monovalent binding of the bispecific antibodygenerated by 2-MEA-induced Fab-arm-exchange betweenIgG1-2F8-F405L×IgG1-7D8-K409R. Nonetheless, the bispecific antibodystill induced CDC-mediated cell kill of the CD20-expressing cells. Thesedata indicate that CDC capacity of a parental antibody was retained inthe bispecific format.

Example 27 ADCC-Mediated Cell Kill by the Bispecific Antibody Generatedby 2-MEA-Induced Fab-Arm-Exchange Between HumanIgG1-2F8-F405L×IgG1-7D8-K409R

The EGFR antibody IgG1-2F8 can kill EGFR-expressing cells, such as A431,by antibody-dependent cellular cytotoxicity (ADCC). A431 cells do notexpress CD20 and therefore the CD20 antibody IgG1-7D8 does not induceADCC on these cells. It was tested whether the mutant IgG1-2F8-F405L andthe bispecific antibody generated by 2-MEA-induced Fab-arm-exchangebetween IgG1-2F8-F405L×IgG1-7D8-K409R were still able to induce ADCC onA431 cells. For effector cell isolation, peripheral blood mononuclearcells (PBMCs) were isolated from whole blood of a healthy donor usingLeucosep® tubes (Greiner Bio-one, cat.#227290) according to themanufacturer's recommendations. Target cells were labelled by adding 100μCi ⁵¹Cr to 5×10⁶ A431 cells in 1 mL RPMI medium supplemented with 0.1%BSA and incubating for 60 min in a 37° C. shaking water bath. Labelledcells were washed and resuspended in RPMI supplemented with 0.1% BSA.5×10⁴ labelled target cells in RPMI supplemented with 0.1% BSA werepreincubated in 100 μL for 15 min with the antibody concentrationsseries (range 0-10 μg/mL final concentration in ADCC assay in 3-folddilutions) at room temperature. The ADCC assay was started by adding 50μL effector cells (5×10⁶ cells) in an E:T ratio 100:1. After 4 hours at37° C., ⁵¹Cr release from triplicate experiments was measured in ascintillation counter as counts per min (cpm). The percentage ofcellular toxicity was calculated using the following formula: percentageof specific lysis=(experimental cpm−basal cpm)/(maximal cpm−basalcpm)×100. Maximal ⁵¹Cr release was determined by adding 50 μL 5% TritonX-100 to 50 μL target cells (5×10⁴ cells), and basal release wasmeasured in the absence of sensitizing antibody and effector cells.

FIG. 22 shows that the CD20-specific antibody IgG1-7D8 did not induceADCC on the CD20-negative A431 cells. Both IgG1-2F8 and the mutantIgG1-2F8-F405L were able to induce ADCC on A431 cells, indicating thatintroduction of the F405L mutation in IgG1-2F8 did not affect its ADCCeffector function. Also the bispecific antibody derived fromIgG1-2F8-F405L×IgG1-7D8-K409R induced ADCC on A431 cells in adose-dependent manner, indicating that the ADCC effector function wasretained in the bispecific format.

Example 28 Determinants at the IgG1 405 Position for Engagement in2-MEA-Induced Fab-Arm-Exchange in Combination with IgG1-K409R

In Example 16 it is described that the F405L mutation is sufficient toenable human IgG1 to engage in Fab-arm-exchange when combined withIgG4-7D8. To further test the determinants at the IgG1 405 position forengagement in 2-MEA-induced Fab-arm-exchange in combination with humanIgG1-K409R, all possible IgG1-2F8-F405X mutants (with the exception of Cand P) were combined with IgG1-7D8-K409R. The procedure was performedwith purified antibodies as described in Example 19.

FIG. 23 shows the results of bispecific binding upon 2-MEA-inducedFab-arm-exchange between IgG1-2F8-F405X×IgG1-7D8-K409R. These data werealso scored as (−) no Fab-arm exchange, (+/−) low, (+) intermediate or(++) high Fab-arm exchange, as presented in Table 3. No Fab-arm exchange(−) was found when the 405 position in IgG1-2F8 was F (=wild type IgG1).Fab-arm exchange was found to be low (+/−) when the 405 position inIgG1-2F8 was G or R. Fab-arm exchange was found to be high (++) when the405 position in IgG1-2F8 was A, D, E, H, I, K, L, M, N, Q, S, T, V, W orY. These data indicate that particular mutations at the IgG1 405position allow IgG1 to engage in 2-MEA-induced Fab-arm-exchange whencombined with IgG1-K409R.

TABLE 3 2-MEA-induced Fab-arm-exchange between IgG1-2F8-F405X mutantsand IgG1-7D8-K409R. Fab-arm-exchange IgG1-2F8-F405X x IgG1-7D8-K409R A++ D ++ E ++ F − G +/− H ++ I ++ K ++ L ++ M ++ N ++ Q ++ R +/− S ++ T++ V ++ W ++ Y ++ The generation of bispecific antibodies after2-MEA-induced in vitro Fab-arm-exchange between IgG1-2F8-F405X mutantsand IgG1-7D8-K409R was determined by a sandwich ELISA. (−) no, (+/−)low, (+) intermediate, (++) high Fab-arm-exchange.

Example 29 Determinants at the IgG1 407 Position for Engagement in2-MEA-Induced Fab-Arm-Exchange in Combination with IgG1-K409R

In Example 28, it is described that certain single mutations at positionF405 are sufficient to enable human IgG1 to engage in Fab-arm-exchangewhen combined with IgG1-K409R. To test whether other determinantsimplicated in the Fc:Fc interface positions in the CH3 domain could alsomediate the Fab-arm-exchange mechanism, mutagenesis of the IgG1 407position was performed and the mutants were tested for engagement in2-MEA-induced Fab-arm-exchange in combination with human IgG1-K409R. Allpossible IgG1-2F8-Y407X mutants (with the exception of C and P) werecombined with IgG1-7D8-K409R. The procedure was performed with purifiedantibodies as described in Example 19.

FIG. 24 shows the results of bispecific binding upon 2-MEA-inducedFab-arm-exchange between IgG1-2F8-Y407X×IgG1-7D8-K409R. These data werealso scored as (−) no Fab-arm exchange, (+/−) low, (+) intermediate or(++) high Fab-arm exchange, as presented in Table 4. No Fab-arm exchange(−) was found when the 407 position in IgG1-2F8 was Y (=wild type IgG1),E, K, Q, or R. Fab-arm exchange was found to be low (+/−) when the 407position in IgG1-2F8 was D, F, I, S or T and intermediate (+) when the407 position in IgG1-2F8 was A, H, N or V, and high (++) when the 407position in IgG1-2F8 was G, L, M or W. These data indicate thatparticular single mutations at the IgG1 407 position allow IgG1 toengage in 2-MEA-induced Fab-arm-exchange when combined with IgG1-K409R.

TABLE 4 2-MEA-induced Fab-arm-exchange between IgG1-2F8-Y407X mutantsand IgG1-7D8-K409R Fab-arm-exchange IgG1-2F8-Y407X x IgG1-7D8-K409R A +D +/− E − F +/− G ++ H + I +/− K − L ++ M ++ N + Q − R − S +/− T +/− V +W ++ Y − The generation of bispecific antibodies after 2-MEA-induced invitro Fab-arm exchange between IgG1-2F8-Y407X mutants and IgG1-7D8-K409Rwas determined by a sandwich ELISA. (−) no, (+/−) low, (+) intermediate,(++) high Fab-arm-exchange.

Example 30 Quantification of the Non-Covalent CH3-CH3 Interaction inIgG1 Heterodimers

It is described in Example 21 that there is a specific range in thestrength of the interaction of the CH3-CH3 homodimers that allowsefficient Fab-arm-exchange. The strength of the interactions at the CH3interface should be such that it is possible that both heavy chains inthe parental antibodies (homodimers) dissociate in the Fab-arm-exchangereaction and that they subsequently associate in the heterodimerizationreaction. To generate a stable heterodimer, the strength of theheterodimer interaction should be greater than the strength of thehomodimer interaction, such that it favors heterodimerization overhomodimerization. To confirm this, the strength of the CH3-CH3interaction in the heterodimers was measured and compared to thestrength in the homodimers. The K_(D) of the CH2-CH3 fragments derivedfrom IgG1-K409R, IgG1-F405L and IgG1-ITL homodimers were measured asdescribed in Example 21. For the determination of the K_(D) inheterodimers, CH2-CH3 domain fragments (G1-F405L and G1-ITL) were mixedwith the IgG1Δhinge fragment of IgG1-7D8-K409R, which contain allantibody domains except the hinge. The lack of hinge regions in bothfragments prevented covalent inter-heavy chain disulfide bonds. Thefragments were mixed and analyzed after 24 hours by native massspectrometry as described in Example 21. The K_(D) values of thenon-covalent CH3-CH3 interactions in the indicated CH2-CH3 fragments ormixtures of CH2-CH3 fragments with IgG1Δhinge are presented in Table 5.These data suggest that under the tested conditions, the strength of theheterodimer interaction is greater (lower K_(D)) than the correspondinghomodimer interactions.

TABLE 5 CH2—CH3 construct/ (IgG1Δhinge) Interaction K_(D) (M)G1-F405L/G1-K409R Heterodimer 1.2 × 10⁻⁸ G1-ITL/G1-K409R Heterodimer 1.7× 10⁻⁸ G1-K409R Homodimer 1.1 × 10⁻⁷ G1-F405L Homodimer 8.5 × 10⁻⁷G1-ITL Homodimer 1.2 × 10⁻⁶

Example 31 Biochemical Analysis of a Bispecific Antibody Generated by2-MEA-Induced Fab-Arm Exchange

A batch of bispecific antibody, generated by 2-MEA-induced Fab-armexchange between human IgG1-2F8-F405L×IgG1-7D8-K409R, was purified on aPD-10 desalting column (cat.no. 17-0851-01; GE Healthcare). Next, thepurity of the bispecific product was analyzed by sodium dodecyl sulfatepolyacrylamide gelelectrophoresis (SDS-PAGE), High Performance SizeExclusion Chromatography (HP-SEC), mass spectrometry, HPLC cationexchange chromatography (HPLC-CIEX), capillary isoelectrofocussing(cIEF).

SDS-PAGE was performed under non-reducing (FIG. 25A) and reducing (FIG.25B) conditions as described in Example 15. FIG. 25A show that theantibody sample after 2-MEA induced Fab-arm exchange consists of intactIgG, with a trace of half molecules (H1L1) detectable on the non-reducedgel.

HP-SEC was performed as described in Example 15. FIG. 26(B) and FIG.26(A) show the HP-SEC profiles of the starting materials IgG1-2F8-F405Land IgG1-7D8-K409R, respectively. The mixture (1:1) of both antibodiesand the bispecific product generated by 2-MEA induced Fab-arm exchangebetween IgG1-2F8-F405L×IgG1-7D8-K409R are shown in FIG. 26C and FIG.26D, respectively. In addition, FIG. 26D shows that >99% of the sampleconsists of intact IgG with practically no aggregates formed.

Mass spectrometry (ESI-MS) was performed as described in Example 12.FIG. 27(B) and FIG. 27(A) show the mass spectrometry profiles of thestarting materials IgG1-2F8-F405L and IgG1-7D8-K409R, respectively. Themixture (1:1) of both antibodies and the bispecific product generated by2-MEA induced Fab-arm exchange between IgG1-2F8-F405L×IgG1-7D8-K409R areshown in FIG. 27C and FIG. 27D, respectively. The product in the 2-MEAinduced Fab-arm exchanged sample is 146,159.7 kDa, which perfectlymatches with the bispecific product derived from IgG1-2F8-F405L(146,289.0/2=73,145)×IgG1-7D8-K409R (146,028.0/2=73,014). Moreover, thebispecific antibody product showed a homogenous peak, indicating that nolight chain mispairing occurred, which would have resulted in subdividedpeaks. These data show that 2-MEA induced Fab-arm exchange resulted inbispecific IgG. The small peaks indicated by (*) resulted fromincomplete deglycosylation prior to analysis. These data show that abispecific antibody sample was generated by 2-MEA-induced Fab-armexchange between IgG1-2F8-F405L×IgG1-7D8-K409R.

Capillary isoelectrofocussing (cIEF) was performed using an iCE280Analyzer (Convergent Biosciences). FIG. 28A and FIG. 28B shows cIEFprofiles of the starting materials IgG1-2F8-F405L and IgG1-7D8-K409R,respectively. The mixture (1:1) of both antibodies and the bispecificproduct generated by Fab-arm exchange betweenIgG1-2F8-F405L×IgG1-7D8-K409R are shown in FIG. 28C and FIG. 28D,respectively. All samples were desalted before use. Final concentrationsin the assay mix were 0.3 mg/mL IgG (0.35% Methyl Cellulose; 2% CarrierAmpholytes 3-10; 6% Carrier Ampholytes 8-10.5; 0.5% pI marker 7.65 and0.5% pI marker 10.10). Focusing was performed for 7 min at 3000 V andthe whole-capillary absorption image was captured by a charge-coupleddevice camera. After calibration of the peak profiles, the data wereanalyzed by the EZChrom software. pI markers are indicated by (*). Thesedata show that a bispecific antibody sample was generated by2-MEA-induced Fab-arm exchange between IgG1-2F8-F405L×IgG1-7D8-K409R.

Another technique to study the charged isoforms of monoclonal antibodiesis High Pressure Liquid Chromatography Cation Exchange (HPLC-CIEX). FIG.29A and FIG. 29B show HPLC-CIEX profiles of the starting materialsIgG1-2F8-F405L and IgG1-7D8-K409R, respectively. The mixture (1:1) ofboth antibodies and the bispecific product generated by 2-MEA inducedFab-arm exchange between IgG1-2F8-F405L×IgG1-7D8-K409R are shown in FIG.29C and FIG. 29D, respectively. Samples were diluted to 1 mg/mL inmobile Phase A (10 mM NaPO4, pH 7.0) for injection onto the HPLC. Thedifferently charged IgG molecules were separated by using a ProPac®WCX-10, 4 mm×250 mm, analytical column with a flow rate of 1 mL/min.Elution was performed with a gradient of Mobile Phase A to Mobile PhaseB (10 mM NaPO₄, pH 7.0, 0.25 M NaCl) and detection occurred at 280 nm.These data show that a bispecific antibody sample was generated by2-MEA-induced Fab-arm exchange between IgG1-2F8-F405L×IgG1-7D8-K409R. Italso shows that cation exchange is a powerful tool to separate residualhomodimers from the heterodimer. Another application of cation exchangechromatography is therefore the polishing of a bispecific heterodimer,i.e. to purify away any residual homodimers after exchange.

Example 32 Recombinant Expression of Heterodimers by SimultaneousCo-Expression of Both Homodimers

To illustrate that heterodimer formation also occurs when the twohomodimers are co-expressed recombinantly, HEK-293F cells wereco-transfected with the four expression vectors (see Example 1) encodingthe heavy and light chain of IgG1-7D8-K409R and IgG1-2F8-F405 in a1:1:1:1 ratio. Antibodies were transiently produced under serum-freeconditions as described in Example 4. Next, IgG was purified by ProteinA chromatography as described in Example 5. Purified IgG wasdeglycosylated and subsequently analyzed by electrospray ionization massspectrometry as described in Example 12.

The theoretic mass of heavy and light chain of IgG1-7D8-K409R andIgG1-2F8-F405 are shown in Table 6.

TABLE 6 Theoretical mass of the heavy and light chain of IgG1-7D8-K409Rand IgG1-2F8-F405 Homodimer L-chain (Da) H-Chain (Da) IgG1-2F8-F40523252.8 49894.6 IgG1-7D8-K409R 23438.1 49579.0

On the basis of these masses, the following IgG molecules couldtheoretically be detected (Table 7). The measured masses (FIG. 30) areindicated in the final column.

TABLE 7 Theoretical detection of heavy and light chain of IgG1-7D8-K409Rand IgG1-2F8-F40 Theoretic Mass IgG1-2F8-F405 IgG1-7D8-K409R massmeasured H-chain L-chain H-chain L-chain (Da) (Da) 2 2 146287 146284 2 2146026 146026 2 2 146657 146664 2 2 145656 145660 2 1 1 146472 146477 12 1 145841 145846 1 1 1 1 146157 146159 1 2 1 145971 145972 1 1 2 146342146345

The two most abundant peaks of 146345 and 146159 Da representedheterodimers with a single (from IgG1-7D8-K409R) or both light chainsincorporated, respectively. Homodimers of both the heavy chain ofIgG1-7D8-K409R or IgG1-2F8-F405 were detected, but only in minoramounts. These data show that heterodimerization also occurs when thetwo homodimers are co-expressed.

Example 33 Monitoring the Kinetics of 2-MEA-Induced Fab-Arm Exchange andQuantifying Residual Homodimers after Exchange by Using HPLC-CIEX

The generation of bispecific antibodies by 2-MEA-induced Fab-armexchange is described in Example 11. In this example the exchangereaction was monitored by conducting High Pressure Liquid ChromatographyCation Exchange (HPLC-CIEX; as described in Example 31) at various timepoints during the exchange reaction.

Homodimers IgG1-2F8-F405L and IgG1-7D8-K409R were mixed in the molarratio 1:1 at a concentration of 1 mg/mL each. After the addition of 25mM 2-MEA, the sample was placed in the autosampler of the HPLC,prewarmed at 25° C. FIG. 31A to 31H shows eight consecutive injectionsat different time intervals obtained by HPLC-CIEX ranging from t=0 minto t=450 min, respectively, after the addition of 2-MEA. The data showthat bispecific IgG was formed rather quickly and most of the homodimerwas exchanged after 135 min. The heterogeneous heterodimer peaksappearing after 45 min resolved into more homogeneous peaks afterapproximately 180 min, suggesting that exchange occurs in differentphases. Furthermore, FIG. 32A shows that approximately 3% of residualhomodimers was detected with the CIEX method (indicated by arrows). Asshown this method is suitable for quantitating the remaining homodimercontent (elution of the homodimers is shown in FIG. 32B) when exchangereaction was almost complete).

Example 34 Generation of Bispecific Antibodies by 2-MEA-Induced Fab-ArmExchange at High Antibody Concentrations at Various 2-MEAConcentrations, Temperatures and Incubation Times

2-MEA induced Fab-arm exchange was performed at high IgG concentrations.The influence of 2-MEA concentration, incubation temperature and time onthe amount of exchange was studied.

The exchange process was performed using the combination ofIgG1-7D8-K409R×IgG1-2F8-F405L. Both materials were purified withaffinity chromatography using protein A. After concentration of thematerial to >20 mg/mL, a successive anion exchange step was performed(in flow through mode) using HiPrep Q FF 16/10 (GE Health Care,#28-9365-43). The final purified material was buffer-exchanged to PBS.

The bispecific exchange was studied at final IgG concentrations of 20mg/mL (each homodimer at a final concentration of 10 mg/mL) and 10 mg/mL(each homodimer at a final concentration of 5 mg/mL) in PBS. Separatemixtures were prepared for both IgG concentrations including 2-MEA atfinal concentrations of 10, 25, 50 and 100 mM. The mixtures were dividedinto 100 μL aliquots in eppendorf tubes and stored at 15, 25 and 37° C.Separate tubes were used for different incubation times of 90 min, 5hours and 24 hours at each temperature.

The mixture was also prepared without 2-MEA for both IgG concentrationsand stored at 4° C. as an untreated control. After the appropriateincubation times, the 90 min and 5 hours samples were collected fordesalting to remove the 2-MEA (the 90 min samples were initially put onice to stop the exchange reaction). The samples were desalted using aZeba 96-well desalting plate (7 k, cat#89808, Thermo Fisher Scientific).The 24 hours samples were desalted separately after 24 hours incubation.

Serial dilutions of the antibody samples (total antibody concentration10-0.123 μg/mL in 3-fold dilutions for the 90 min and 5 hours samples;10-0.041 μg/mL in 3-fold dilutions for the 24 hours samples) were usedin a sandwich ELISA to measure bispecific binding as described inExample 7. For each plate, a control was included of a purified batch ofbispecific antibody derived from a 2-MEA-induced Fab-arm exchangebetween IgG1-2F8-ITL and IgG4-7D8-CPPC (as described in Example 15).FIG. 34(A)-(F) shows the results of the bispecific binding as measuredin the individual ELISA plates. The top OD405 values (as determined forthe 10 μg/mL concentrations in the ELISA) were used to calculate thebispecific binding in comparison to the control, which was arbitrarilyset at 100%. This resulted in the percentage of controlled Fab-armexchange (% cFAE) compared to the control as is shown in FIG. 34(A)-(D)for each 2-MEA concentration.

The data show that maximal level of bispecific binding (89-109% withrespect to control) was reached at a concentration of 100 mM 2-MEA forboth IgG concentrations at all temperature-time conditions. At 50 mM2-MEA, maximal binding (88-107%) was achieved at 25° C. and 37° C. andalso at 15° C. after 24 hours incubation. For the lower concentrationsof 25 mM and 10 mM 2-MEA, the exchange was more efficient at highertemperatures and increased with prolonged incubation time, leading tomaximal exchange at 37° C. upon 24 hours incubation at 25 mM 2-MEA. Noneof the conditions tested at 10 mM 2-MEA generated 100% bispecificproduct. The exchange process was slightly faster at IgG concentrationsof 10 mg/mL compared to 20 mg/mL total IgG.

To confirm that bispecific antibodies were formed and to study thebispecific products in more detail, samples were analyzed with CationExchange (HPLC-CIEX) analysis. The HPLC-CIEX analysis was performed asdescribed in Example 31 for the samples with IgG concentrations of 20mg/mL after 5 hours and 24 hours incubation and all 2-MEAconcentrations.

The CIEX chromatograms in FIG. 35(A)-(D) show that the highest yield ofbispecific product was obtained at 50 and 100 mM 2-MEA confirming theresults of the bispecific ELISA. However, minor amounts of residualhomodimer were still detected at 50 and 100 mM 2-MEA (2-3.5% of eachhomodimer for samples incubated at 25° C. and 37° C.). Exchange athigher temperature, longer (24 hours) incubation time and increasing2-MEA concentration result in the appearance of additional peaks at22-24 min in the CIEX profile.

Minimal amounts of additional peaks were obtained when exchange wasconcluded within 5 hours. To identify the nature of these peaks,SDS-PAGE analysis and HP-SEC analysis was performed. HP-SEC showed thatthe amount of aggregates was below 1% for all conditions, suggestingthat the additional peaks do not represent aggregates. However,non-reduced SDS-PAGE indicated that the extra peaks may representheterodimer lacking one or two light chains. Minor amounts ofhalf-molecules were detected as well.

The experiment shows that the exchange reaction takes place at highhomodimer concentrations, which makes the process attractive forcommercial scale, and that the yield of bispecific antibody depends on2-MEA concentration, temperature and incubation time.

Example 35 Determinants at the IgG1 368 Position for Engagement in2-MEA-Induced Fab-Arm Exchange in Combination with IgG1-K409R

Example 28 and 29 show that certain single mutations at position F405and Y407 are sufficient to enable human IgG1 to engage in Fab-armexchange when combined with IgG1-K409R. As illustrated in this examplefurther determinants implicated in the Fc:Fc interface positions in theCH3 domain may also mediate the Fab-arm exchange mechanism. To thiseffect mutagenesis of the IgG1 368 position was performed and themutants were tested for engagement in 2-MEA-induced Fab-arm-exchange incombination with human IgG1-K409R. All possible IgG1-2F8-L368X mutants(with the exception of C and P) were combined with IgG1-7D8-K409R. Theprocedure was performed with purified antibodies as described in Example19.

FIG. 36 shows the results of bispecific binding upon 2-MEA-inducedFab-arm exchange between IgG1-2F8-L368X×IgG1-7D8-K409R. These data werealso scored as (−) no Fab-arm exchange, (+/−) low, (+) intermediate or(++) high Fab-arm exchange, as presented in Table 8. No Fab-arm exchange(−) was found when the 368 position in IgG1-2F8 was L (=wild type IgG1),F or M. Fab-arm exchange was found to be low (+/−) when the 368 positionin IgG1-2F8 was Y. Fab-arm exchange was found to be intermediate (+)when the 368 position in IgG1-2F8 was K and high (++) when the 368position in IgG1-2F8 was A, D, E, G, H, I, N, Q, R, S, T, V, or W. Thesedata indicate that particular mutations at the IgG1 368 position allowIgG1 to engage in 2-MEA-induced Fab-arm exchange when combined withIgG1-K409R.

TABLE 8 2-MEA-induced Fab-arm exchange between IgG1-2F8-L368X mutantsand IgG1-7D8-K409R Fab-arm exchange Fab-arm exchange IgG1-2F8-L368X xIgG1-7D8-K409R A ++ D ++ E ++ F − G ++ H ++ I ++ K + L − M − N ++ Q ++ R++ S ++ T ++ V ++ W ++ The generation of bispecific antibodies after2-MEA-induced in vitro Fab-arm exchange between IgG1-2F8-L368X mutantsand IgG1-7D8-K409R was determined by a sandwich ELISA. (−) no, (+/−)low, (+) intermediate or (++) high Fab-arm exchange.

Example 36 Determinants at the IgG1 370 Position for Engagement in2-MEA-Induced Fab-Arm Exchange in Combination with IgG1-K409R

Examples 28, 29 and 35 show that certain single mutations at positionsF405, Y407 or L368 are sufficient to enable human IgG1 to engage inFab-arm exchange when combined with IgG1-K409R. As illustrated in thisexample further determinants implicated in the Fc:Fc interface positionsin the CH3 domain may also mediate the Fab-arm exchange mechanism. Tothis effect mutagenesis of the IgG1 370 position was performed and themutants were tested for engagement in 2-MEA-induced Fab-arm-exchange incombination with human IgG1-K409R. All possible IgG1-2F8-K370X mutants(with the exception of C and P) were combined with IgG1-7D8-K409R. Theprocedure was performed with purified antibodies as described in Example19.

FIG. 37 shows the results of bispecific binding upon 2-MEA-inducedFab-arm exchange between IgG1-2F8-K370X×IgG1-7D8-K409R. These data werealso scored as (−) no Fab-arm exchange, (+/−) low, (+) intermediate or(++) high Fab-arm exchange, as presented in Table 9. No Fab-arm exchange(−) was found when the 370 position in IgG1-2F8 was K (=wild type IgG1),A, D, E, F, G, H, I, L, M, N, Q, R, S, T, V or Y. Only substitution ofK370 with W resulted in intermediate Fab-arm exchange (+). These dataindicate that only one mutation at the IgG1 370 position (K370W) allowsIgG1 to engage in 2-MEA-induced Fab-arm exchange when combined withIgG1-K409R.

TABLE 9 2-MEA-induced Fab-arm exchange between IgG1-2F8-K370X mutantsand IgG1-7D8-K409R Fab-arm exchange IgG1-2F8-K370X x IgG1-7D8-K409R A −D − E − F − G − H − I − K − L − M − N − Q − R − S − T − V − W + Y − Thegeneration of bispecific antibodies after 2-MEA-induced in vitro Fab-armexchange between IgG1-2F8-K370X mutants and IgG1-7D8-K409R wasdetermined by a sandwich ELISA. (−) no, (+/−) low, (+) intermediate or(++) high Fab-arm exchange.

Example 37 Determinants at the IgG1 399 Position for Engagement in2-MEA-Induced Fab-Arm Exchange in Combination with IgG1-K409R

Examples 28, 29, 35 and 36 show that certain single mutations atpositions F405, Y407, L368 or K370 are sufficient to enable human IgG1to engage in Fab-arm exchange when combined with IgG1-K409R. Asillustrated in this example further determinants implicated in the Fc:Fcinterface positions in the CH3 domain may also mediate the Fab-armexchange mechanism. To this effect mutagenesis of the IgG1 399 positionwas performed and the mutants were tested for engagement in2-MEA-induced Fab-arm-exchange in combination with human IgG1-K409R. Allpossible IgG1-2F8-D399X mutants (with the exception of C and P) werecombined with IgG1-7D8-K409R. The procedure was performed with purifiedantibodies as described in Example 19.

FIG. 38 shows the results of bispecific binding upon 2-MEA-inducedFab-arm exchange between IgG1-2F8-D399X×IgG1-7D8-K409R. These data werealso scored as (−) no, (+/−) low, (+) intermediate or (++) high Fab-armexchange, as presented in Table 10. No Fab-arm exchange (−) was foundwhen the 399 position in IgG1-2F8 was D (=wild type IgG1), E and Q.Fab-arm exchange was found to be low (+/−) when the 399 position inIgG1-2F8 was V, intermediate (+) when the 399 position in IgG1-2F8 wasG, I, L, M, N, S, T or W. Fab-arm exchange was found to be high (++)when the 399 position in IgG1-2F8 was A, F, H, K, R or Y. These dataindicate that particular mutations at the IgG1 399 position allow IgG1to engage in 2-MEA-induced Fab-arm exchange when combined withIgG1-K409R.

TABLE 10 2-MEA-induced Fab-arm exchange between IgG1-2F8-D399X mutantsand IgG1-7D8-K409R Fab-arm exchange IgG1-2F8-D399X x IgG1-7D8-K409R A ++D − E − F ++ G + H ++ I + K ++ L + M + N + Q − R ++ S + T + V +/− W + Y++ The generation of bispecific antibodies after 2-MEA-induced in vitroFab-arm exchange between IgG1-2F8-D399X mutants and IgG1-7D8-K409R wasdetermined by a sandwich ELISA. (−) no, (+/−) low, (+) intermediate or(++) high Fab-arm exchange.

Example 38 Determination of the Condition Range in which 2-MEA-InducedFab-Arm Exchange Occurs Suboptimally to Discriminate Between HighlyEfficient IgG1 Mutants

The process of 2-MEA-induced Fab-arm exchange occurs efficiently at 37°C. when 25 mM 2-MEA is used. Under these conditions, the majority ofpermissive IgG1 mutants (IgG1 with certain single mutations at positions368, 370, 399, 405 and 407 and/or 409 as described in Examples 19, 28,29, and 35-37) show high levels of 2-MEA-induced Fab-arm exchange(80%-100%). To identify experimental conditions that would allowdiscrimination between the IgG1 mutants with the highest efficiency,2-MEA-induced Fab-arm for four different mutant combinations(IgG1-2F8-F405S×IgG1-7D8-K409A, IgG1-2F8-D399R×IgG1-7D8-K409G,IgG1-2F8-L368R×IgG1-7D8-K409H and IgG1-2F8-F405L×IgG1-7D8-K409R) wasstudied over time at 15° C. and 20° C., respectively. Apart from changesin temperature, time period and antibody dilution (20, 2, 0.2 and 0.02μg/mL) the procedure was performed as described in Example 19.

At 20° C. 2-MEA-induced Fab-arm exchange of the four mutant combinationsoccurred at different rates compared to the maximal exchange (positivecontrol). After 105 min incubation IgG1-2F8-L368R×IgG1-7D8-K409H reachedthe maximal level of exchange, whereas IgG1-2F8-F405S×IgG1-7D8-K409A,IgG1-2F8-D399R×IgG1-7D8-K409G and IgG1-2F8-F405L×IgG1-7D8-K409R reacheda maximum of 90%, 85% and 85%, respectively, after 200 min.

Incubation of the different IgG1 mutant combinations at 15° C. showedmost prominent differences in exchange rates (shown in FIG. 39). After60 and 105 min incubations, 2-MEA-induced Fab-arm exchange, thedifferences between the four mutant combinations were most extreme.Fab-arm exchange after 200 min incubation showed efficiencies of 100%(IgG1-2F8-L368R×IgG1-7D8-K409H), 85% (IgG1-2F8-F405L×IgG1-7D8-K409R andIgG1-2F8-D399R×IgG1-7D8-K409G) or 65% (IgG1-2F8-F405S×IgG1-7D8-K409A)compared to the positive control.

Example 39 Analyzing 2-MEA-Induced Fab-Arm Exchange Efficiencies ofMutants at Suboptimal Conditions

The process of 2-MEA-induced Fab-arm exchange occurs efficiently at 37°C. when 25 mM 2-MEA is used. Under these conditions, the majority ofpermissive IgG1 mutants (IgG1 with certain single mutations at positions368, 370, 399, 405 and 407 and/or 409 as described in Examples 19, 28,29, and 35-37) show high levels of 2-MEA-induced Fab-arm exchange(80-100%). In Example 38 it is described that differences in2-MEA-induced Fab-arm exchange efficiencies are most pronounced afterincubation at so called suboptimal conditions, namely at 15° C. for 60to 105 min. In total 24 IgG1-2F8 mutants at the L368, D399, F405 andY407 (see Table 11) that show >90% 2-MEA-induced Fab-arm exchange withIgG1-7D8-K409R (Example 28, 29, and 35-37) were selected and subjectedto Fab-arm exchange analysis with IgG1-7D8-K409A, G, H or R (based onresults reported in Example 19). To categorize these mutant combinationsupon their efficiencies to generate bispecific antibodies, 2-MEA-inducedFab-arm exchange was performed at 15° C. for 90 min (suboptimalconditions). Two IgG1-2F8 mutants Y407Q and D399Q) that showed weak2-MEA-induced Fab-arm exchange after incubation with IgG1-7D-K409R(Example 29 and 37) were taken along as additional negative controls andused to study whether incubation with another amino acid at the K409position (G, H, or W) leads to a different result. Apart from a changein temperature and changes in antibody dilution (20, 2, 0.2 and 0.02ug/mL), the procedure was performed as described in Example 19.

Incubation of all different IgG1 mutants combinations (as becomes clearfrom Table 11) at 15° C. for 90 min showed a range of different2-MEA-induced Fab-arm exchange efficiencies. The result of bispecificbinding at an antibody concentration of 20 μg/mL, is shown in Table 11.Results were categorized in 4 classes; no (−), low (+/−) intermediate(+) and high (++) bispecific binding efficiency as is specified in thelegend below for Table 11. From these results it becomes clear thatunder suboptimal conditions some combinations of amino acid mutations inIgG1 molecules will be favorable for 2-MEA-induced Fab-arm exchange.

TABLE 11 Bispecific binding (% relative to positive control) betweenpermissive IgG1 mutants (20 μg/mL) at 15° C. for 90 min

From the mutated IgG1-2F8 molecules tested (Table 11), six were selectedfor a second analysis to confirm the results obtained before (Table 11).Several mutants were selected for their high (IgG1-2F8-L368R) andintermediate (IgG1-2F8-L368W, IgG1-2F8-F4051, IgG1-2F8-F405L andIgG1-2F8-Y407W) 2-MEA-induced Fab-arm exchange efficiency. AlsoIgG1-2F8-Y407Q was analyzed for a second time since it showed anunexpected positive 2-MEA-induced Fab-arm exchange reaction withIgG1-7D8-K409H. In general, these results, presented in FIG. 40,confirmed the primary analysis (Table 11) and show that 2-MEA-inducedFab-arm exchange reactions of mutated IgG1-2F8 molecules withIgG1-7D8-K409H showed highest efficiency. Furthermore, 2-MEA-inducedFab-arm exchange reactions between mutated IgG1-2F8 molecules withIgG1-7D8-K409R that are reported in Examples 28, 29, and 35-37 asnegative are still of interest as potentially promoting the IgG12-MEA-induced Fab-arm exchange.

Example 40 Using the Bispecific Format to Remove Undesired AgonisticActivity of Antagonistic c-Met Antibodies Converting them into aMonovalent, Bispecific Format

Several bivalent antibodies developed for monoclonal antibody therapyshow undesirable agonistic activity upon binding to their target. Thisis also the case for most IgG1 based antibodies targeting the receptortyrosine kinase c-Met. These agonistic antibodies induce receptordimerization followed by activation of several downstream signalingpathways. As a result growth and differentiation of (tumor) cells isinduced. The use of monovalent antibody formats can prevent induction ofreceptor dimerization. Combination of an anti-c-Met antibody Fab-armwith a Fab-arm of an irrelevant antibody results in a bispecificantibody that is functionally monovalent and therefore completelyantagonistic. Here we combined a partial-(IgG1-069) or a full (IgG1-058)agonistic antibody, with IgG1-b12 (first described in Burton D R, et al,“Efficient neutralization of primary isolates of HIV-1 by a recombinanthuman monoclonal antibody”, Science. 1994 Nov. 11; 266(5187):1024-1027)in bispecific antibodies. IgG1-b12 was regarded as an irrelevantnon-binding antibody since it is raised against a viral protein(HIV-gp120). The anti-c-Met antibodies used in this example are fullyhuman monoclonal antibodies generated in transgenic mice. IgG1-058 andIgG1-069 bind to different epitopes on c-Met.

The two anti-c-Met antibodies used are IgG1,κ antibodies being modifiedin their Fc regions as further disclosed. They have the following heavychain and light chain variable sequences.

058: VH 058 EVQLVESGGGLVKPGGSLKLSCAASGFTFSDYYMYWVRQTPEKRLEWVATISDDGSYTYYPDSVKGRFTISRDNAKNNLYLQMSSLKSEDTAMYYCAREGLYYYGSGSYYNQDYWGQGTLVTV SS VL 058AIQLTQSPSSLSASVGDRVTITCRASQGLSSALAWYRQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPED FATYYCQQFTSYPQITFGQGTRLEIK069: VH 069 QVQLVQSGAEVKKPGASVKVSCETSGYTFTSYGISWVRQAPGHGLEWMGWISAYNGYTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARDLRGTNYFDYWGQGTLVTVSS VL 069DIQMTQSPSSVSASVGDRVTITCRASQGISNWLAWFQHKPGKAPKLLIYAASSLLSGVPSRFSGSGSGTDFTLTISSLQPED FATYYCQQANSFPITFGQGTRLEIK

Receptor Phosphorylation

Monovalent bispecific c-Met antibodies were generated by a Fab-armexchange reaction with IgG1-058-F405L or IgG1-069-F405L andIgG1-b12-K409R as described in Example 23 using 25 mM 2-MEA. The effectof the bispecific antibody on c-Met phosphorylation was evaluated. Upondimerization of two adjacent c-Met receptors by either the naturalligand HGF or by agonistic bivalent antibodies, three tyrosine residues(position 1230, 1234 and 1235) in the intracellular domain of c-Met arecross phosphorylated. This leads to phosphorylation of several otheramino acids in the intracellular domain of c-Met and activation of anumber of signaling cascades. The dimerization and activation of c-Metcan be monitored by using antibodies specific for the phosphorylatedreceptor at these positions, which functions as a read out for thepotential agonism of anti-c-Met antibodies.

A549 cells, CCL-185 obtained from ATCC, were grown in serum containingDMEM medium until 70% confluency was reached. Cells were trypsinized,washed and plated in a 6 well culture plate at 1*10e6 cells/well inserum containing culture medium. After overnight incubation the cellswere treated with either HGF (R&D systems; cat. 294-HG) (50 ng/mL) orthe panel of antibodies (30 μg/mL) and incubated for 15 minutes at 37°C. The cells were washed twice with ice cold PBS and lysed with lysisbuffer (Cell Signaling; cat. 9803) supplemented with a proteaseinhibitor cocktail (Roche; cat. 11836170001). Cell lysate samples werestored at −80° C. Receptor activation was determined by detection ofc-Met phosphorylation on Western blot using phospho c-Met specificantibodies. The proteins present in the cell lysate were separated on a4-12% SDS-PAGE gel and transferred to nitrocellulose membrane that wassubsequently stained with an antibody specific for phosphorylated c-Met(Y1234/1235) (Cell Signaling, cat: 3129). As a control for gel loadingtotal β-actin and c-Met levels were determined using anti c-Met (CellSignaling, Cat. No. 3127) and anti β-actin (Cell Signaling, Cat. No.4967) antibodies. Results of the Western blots are shown in FIG. 41.

Tissue culture medium controls and cells treated with the monovalentformat UniBody® (Genmab, WO2007059782 and WO2010063785) of antibody 5D5(Genentech; WO 96/38557) did not show any c-Met receptorphosphorylation. The monovalent UniBody format as used herein is anIgG4, wherein the hinge region has been deleted and wherein the CH3region has been mutated at positions 405 and 407. In contrast, Westernblot analysis of cells treated with the positive control HGF oragonistic antibody IgG1-058 showed a clear band at the expected heightof the phosphorylated c-Met. Partial agonistic antibody IgG1-069 showedless, but detectable receptor phosphorylation indicating that some crosslinking of the receptor takes place. However, both bispecific IgG1058/b12 and bispecific 069/b12 antibodies did not induce c-Metphosphorylation at all, showing that the agonistic activity associatedwith the parent antibodies was completely absent (FIG. 41).

Effect of c-Met Antibodies on NCI-H441 Proliferation In Vitro

The potential proliferative agonistic activity of c-Met antibodies wastested in vitro in the lung adenocarcinoma cell line NCI-H441 (ATCC,HTB-174™). This cell line expresses high levels of c-Met, but does notproduce its ligand HGF. NCI-H441 cells were seeded in a 96-wells tissueculture plate (Greiner bio-one, Frickenhausen, Germany) (5,000cells/well) in RPMI (Lonza) without serum. Anti c-Met antibody wasdiluted to 66.7 nM in RPMI medium without serum and added to the cells.After seven days incubation at 37° C./5% CO₂, the amount of viable cellswas quantified with Alamarblue (BioSource International, San Francisco,US) according to the manufacturer's instruction. Fluorescence wasmonitored using the EnVision 2101 Multilabel reader (PerkinElmer, Turku,Finland) with standard Alamarblue settings.

In contradiction to IgG1-069, no proliferation was induced uponincubation of NCI-H441 cells with the bispecific IgG1-069/b12, as isshown in FIG. 42. Also the UniBody-069 control did not induceproliferation, which was comparable to the none- or IgG1-b12 treated.

Example 41 CDC-Mediated Cell Killing by Bispecific Antibodies Generatedby 2-MEA-Induced Fab-Arm-Exchange Between Human IgG1-2F8-F405L orIgG1-7D8-F405L and IgG1-7D8-K409R

The CD20 antibody IgG1-7D8 can efficiently kill CD20-expressing cells bycomplement-dependent cytotoxicity (CDC). In contrast, the EGFR antibodyIgG1-2F8 does not mediate CDC on target cells expressing EGFR. BothIgG1-7D8-K409R and the bispecific antibody generated by 2-MEA-inducedFab-arm-exchange between IgG1-2F8-F405L×IgG1-7D8-K409R are able toinduce CDC on CD20-expressing cells (as is described in Example 26). Itwas tested whether the bispecific antibody generated by 2-MEA-inducedFab-arm-exchange between IgG1-7D8-F405L and IgG1-7D8-K409R could alsoinduce CDC on CD20 expressing cells. 10⁵ Daudi or Raji cells werepre-incubated for 15 min with a concentration series of antibody in 100μL RPMI medium supplemented with 0.1% BSA in a shaker at roomtemperature. 25 μL normal human serum (NHS) was added as a source ofcomplement (20% NHS final concentration) and incubated for 45 min at 37°C. After incubation, plate was placed on ice to stop the CDC reaction.Dead and viable cells were discriminated by adding 10 μL 10 μg/mLpropidium iodide (PI) (0.6 μg/mL final concentration) and FACS analysis.

FIG. 43 shows that IgG1-7D8 and the bispecific product generated by2-MEA-induced Fab-arm-exchange between IgG1-7D8-F405L and IgG1-7D8-K409Rhave the same potency to induce CDC-mediated cell kill ofCD20-expressing Daudi (FIG. 43A) and Raji (FIG. 43B). Both Daudi andRaji cells do not express EGFR, resulting in monovalent binding of thebispecific antibody generated by 2-MEA-induced Fab-arm-exchange betweenIgG2-2F8-F405L×IgG1-7D8-K409R This bispecific product also induced CDCmediated cell kill, albeit slightly less efficient. These data indicatethat CDC capacity of a parental antibody was retained in the bispecificformat. Induction of CDC mediated cell killing by the bivalentbispecific product (IgG1-7D8-F405L×IgG1-7D8-K409R) was slightly moreefficient compared to the monovalent bispecific product(IgG2-2F8-F405L×IgG1-7D8-K409R). The CD20 targeting 11B8 antibody is notable to induce CDC mediated cell kill and functions as a negativecontrol.

Example 42 HER2×HER2 Bispecific Antibodies Tested in an In VitroKappa-Directed ETA′ Killing Assay

The example shows that HER2×HER2 bispecific antibodies can deliver acytotoxic agent into tumor cells after internalization in a generic invitro cell-based killing assay using kappa-directed pseudomonas-exotoxinA (anti-kappa-ETA′). This assay makes use of a high affinity anti-kappadomain antibody conjugated to a truncated form of thepseudomonas-exotoxin A. Similar fusion proteins of antibody bindingproteins (IgG-binding motif from Streptococcal protein A or protein G)and diphtheria toxin or Pseudomonas exotoxin A have previously been(Mazor Y. et al., J. Immunol. Methods 2007; 321:41-59); Kuo S R. et al.,2009 Bioconjugate Chem. 2009; 20:1975-1982). These molecules in contrastto anti-kappa-ETA′ bound the Fc part of complete antibodies. Uponinternalization and endocytic sorting the anti-kappa-ETA′ domainantibody undergoes proteolysis and disulfide-bond reduction, separatingthe catalytic from the binding domain. The catalytic domain is thentransported from the Golgi to the endoplasmic reticulum via a KDELretention motif, and subsequently translocated to the cytosol where itinhibits protein synthesis and induces apoptosis (Kreitman R J. et. al.,BioDrugs 2009; 23:1-13).

The anti-HER2 antibodies used in this example and the following Examples43-45 are fully human monoclonal antibodies generated in transgenicmice. They bind to different epitopes on HER2.

They are all IgG1,κ antibodies being modified in their Fc regions asfurther disclosed. They have the following heavy chain and light chainvariable sequences.

005: VH 005 EVQLVQSGAEVKKPGESLKISCKASGYSFHFYWIGWVRQMPGKGLEWMGSIYPGDSDTRYRPSFQGQVTISADKSISTAYLQWTSLKASDTAIYYCARQRGDYYYFYGMDVWGQGTTVTVSS VL 005EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQVPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAV YYCQQYGSS-LTFGGGTKVEIK 025:VH 025 QVQLQQWGAGLLKPSETLSLTCAVYGGSFSDYYWNWIRQPPGKGLEWIGEIHHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGYYDSGVYYFDYWAQGTLVTVSS VL 025DIQMTQSPSSLSASVGDRVTITCRASQGISRWLAWYQQKPEKAPKSLIYAASSLRSGVPSRFSGSGSGTDFTLTISSLQPEDFATY YCQQYNSYPITFGQGTRLEIK 153:VH 153 QVQLVESGGGVVQPGRSLRLSCAASGFTFSDYVIHWVRQAPGKGLEWVTVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLSAEDTAMYYCARGGITGTTGVFDYWGQGTLVTVSS VL 153DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYDASSLQSGVPSRFSGSGYGTDFSLTISSLQPEDFAIY YCQQYKSYPITFGQGTRLEIK 169:VH 169 QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGISWVRQAPGQGLEWMGWLSAYSGNTIYAQKLQGRVTMTTDTSTTTAYMELRSLRSDDTAVYYCARDRIVVRPDYFDYWGQGTLVTVSS VL 169EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVY YCQQRSNWPRTFGQGTKVEIK

HER2×HER2 bispecific antibodies were pre-incubated with theanti-kappa-ETA′ before incubation with A431 cells. A431 cells express˜15,000 HER2 antibodies per cell (determined via Qifi analysis) and arenot sensitive to treatment with ‘naked’ HER2-antibodies.

First, the optimal concentration of anti-kappa-ETA′ was determined foreach cell line, i.e. the maximally tolerated dose that does not lead toinduction of non-specific cell death. A431 cells (2500 cells/well) wereseeded in normal cell culture medium in a 96-wells tissue culture plate(Greiner bio-one) and allowed to adhere for at least 4 hours. Thesecells were incubated with an anti-kappa-ETA′ dilution series, 100, 10,1, 0.1, 0.01, 0.001 and 0 μg/mL in normal cell culture medium. After 3days, the amount of viable cells was quantified with Alamarblue(BioSource International, San Francisco, US) according to themanufacturer's instruction. Fluorescence was monitored using theEnVision 2101 Multilabel reader (PerkinElmer, Turku, Finland) withstandard Alamarblue settings. The highest concentration anti-kappa-ETA′that did not kill the cells by itself (1 μg/mL for A431 cells) was usedfor following experiments.

Next, the effect of HER2×HER2 bispecific antibodies and HER2monospecific antibodies pre-incubated with anti-kappa-ETA′ was testedfor their ability to induce cell kill. A431 cells were seeded asdescribed above. A dilution series of the HER2 specific antibodies(monospecific and bispecific antibodies) was made and pre-incubated for30 min with the predetermined concentration of anti-kappa-ETA′ beforeadding them to the cells. After 3 days incubation at 37° C., the amountof viable cells was quantified as described above. The Alamarblue signalof cells treated with anti-kappa-ETA′ pre-incubated with the antibodieswas plotted compared to cells treated without antibody treatment. EC₅₀values and maximal cell death were calculated using GraphPad Prism 5software. Staurosporin (23.4 μg/mL) was used as positive control forcell killing. An isotype control antibody (IgG1/kappa; IgG1-3G8-QITL)was used as negative control.

FIG. 44 shows that all anti-kappa-ETA′ pre-incubated HER2 bispecificantibodies were able to kill A431 cells in a dose-dependent manner.These results demonstrate that most HER2 bispecific antibodies testedwere more effective than the monospecific antibody present in thecombination in this anti-kappa-ETA′ assay. In addition, the efficacy ofbispecific antibody 005X169, 025X169 and 153X169 showed that theefficacy of a monospecific antibody which lacks activity in this invitro kappa-directed ETA′ killing, HER2 specific antibody 169, can beincreased through bispecific combination with another HER2 specificantibody.

Example 43 HER2 Receptor Downmodulation by Incubation with BispecificAntibodies Targeting Different HER2 Epitopes

HER2×HER2 bispecific antibodies may bind two different epitopes on twospatially different HER2 receptors. This may allow other HER2×HER2bispecific antibodies to bind to the remaining epitopes on thesereceptors. This could result in multivalent receptor cross-linking(compared to dimerization induced by monovalent antibodies) andconsequently enhance receptor downmodulation. To investigate whetherHER2×HER2 bispecific antibodies induce enhanced downmodulation of HER2,AU565 cells were incubated with antibodies and bispecific antibodies forthree days. Total levels of HER2 and levels of antibody bound HER2 weredetermined.

AU565 cells were seeded in a 24-well tissue culture plate (100.000cells/well) in normal cell culture medium and cultured for three days at37° C. in the presence of 10 μg/mL HER2 antibody or HER2×HER2 bispecificantibodies. After washing with PBS, cells were lysed by incubating themfor 30 min at room temperature with 25 μL Surefire Lysis buffer (PerkinElmer, Turku, Finland). Total protein levels were quantified usingbicinchoninic acid (BCA) protein assay reagent (Pierce) followingmanufacturer's protocol. HER2 protein levels in the lysates wereanalyzed using a HER2-specific sandwich ELISA. Rabbit-anti-human HER2intracellular domain antibody (Cell Signaling) was used to capture HER2and biotinylated goat-anti-human HER2 polyclonal antibody R&D systems,Minneapolis, USA), followed by streptavidin-poly-HRP, were used todetect bound HER2. The reaction was visualized using 2,2′-azino-bis3-ethylbenzothiazoline-6-sulfonic acid (one ABTS tablet diluted in 50 mLABTS buffer [Roche Diagnostics, Almere, The Netherlands]) and stoppedwith oxalic acid (Sigma-Aldrich, Zwijndrecht, The Netherlands).Fluorescence at 405 nm was measured on a microtiter plate reader (BiotekInstruments, Winooski, USA) and the amount of HER2 was expressed as apercentage relative to untreated cells.

The results are shown in FIG. 45 which demonstrates that all the testedHER2×HER2 bispecific antibodies induced ≧0.40% HER2 downmodulation.Interestingly, all HER2×HER2 bispecific antibodies demonstratedincreased HER2 downmodulation compared to both of their monospecificcounterparts.

Example 44 Colocalization of HER2×HER2 Bispecific Antibodies withLysosomal Marker LAMP1 Analyzed by Confocal Microscopy

The HER2 downmodulation assay as described in Example 43 indicated thatHER2×HER2 bispecific antibodies were able to increase lysosomaldegradation of HER2. To confirm these findings, confocal microscopytechnology was applied. AU565 cells were grown glass coverslips(thickness 1.5 micron, Thermo Fisher Scientific, Braunschweig, Germany)in standard tissue culture medium for 3 days at 37° C. Cells werepre-incubated for 1 hour with leupeptin (Sigma) to block lysosomalactivity after which 10 ug/mL HER2 monospecific antibodies or HER2×HER2bispecific antibodies were added. The cells were incubated for anadditional 3 or 18 hours at 37° C. Hereafter they were washed with PBSand incubated for 30 min. at room temperature with 4% formaldehyde(Klinipath). Slides were washed with blocking buffer (PBS supplementedwith 0.1% saponin [Roche] and 2% BSA [Roche]) and incubated for 20 minwith blocking buffer containing 20 mM NH₄Cl to quench formaldehyde.Slides were washed again with blocking buffer and incubated for 45 minat room temperature with mouse-anti-human CD107a (LAMP1) (BD Pharmingen)to stain lysosomes. Following washing with blocking buffer the slideswere incubated 30 min at room temperature with a cocktail of secondaryantibodies; goat-anti-mouse IgG-Cy5 (Jackson) and goat-anti-humanIgG-FITC (Jackson). Slides were washed again with blocking buffer andmounted overnight on microscope slides using 20 μL mounting medium (6gram Glycerol [Sigma] and 2.4 gram Mowiol 4-88 [Omnilabo] was dissolvedin 6 mL distilled water to which 12 mL 0.2M Tris [Sigma] pH8.5 was addedfollowed by incubation for 10 min at 50-60° C. Mounting medium wasaliquoted and stored at −20° C.). Slides were imaged with a Leica SPE-IIconfocal microscope (Leica Microsystems) equipped with a 63×1.32-0.6 oilimmersion objective lens and LAS-AF software. To allow forquantification of overlapping pixel intensities, saturation of pixelsshould be avoided. Therefore the FITC laser intensity was decreased to10%, smart gain was set at 830 V and smart offset was set at −9.48%. Byusing these settings, the bispecific antibodies were clearly visualizedwithout pixel saturation, but the monospecific antibodies were sometimesdifficult to detect. To compare lysosomal colocalization betweenmonospecific and bispecific antibodies, these settings were kept thesame for all analyzed confocal slides.

12-bit images were analyzed for colocalisation using MetaMorph® software(version Meta Series 6.1, Molecular Devices Inc, Sunnyvale Calif., USA).FITC and Cy5 images were imported as stacks and background wassubtracted. Identical thresholds settings were used (manually set) forall FITC images and all Cy5 images. Colocalisation was depicted as thepixel intensity of FITC in the region of overlap (ROI), were the ROI iscomposed of all Cy5 positive regions. To compare different slidesstained with several HER2 antibodies or HER2×HER2 bispecific antibodies,the images were normalized using the pixel intensity of Cy5.Goat-anti-mouse IgG-Cy5 was used to stain the lysosomal marker LAMP1(CD107a). The pixel intensity of LAMP1 should not differ between variousHER2 antibodies or the HER2×HER2 bispecific antibodies tested (one cellhad a pixel intensity of Cy5 of roughly 200.000).

Normalized values for colocalization of FITC andCy5=[(TPI-FITC×percentage FITC-Cy5colocalization)/100]×[200.000/TPI-Cy5]

In this formula, TPI stands for Total Pixel Intensity.

presents percentage of viable cells, as measured by the FITC pixelintensity overlapping with Cy5 for various monospecific HER2 antibodiesand HER2×HER2 bispecific antibodies. For each antibody or bispecificmolecule depicted, three different images were analyzed from one slidecontaining ˜1, 3 or >5 cells. Significant variation was observed betweenthe different images within each slide. However, it was evident that allHER2×HER2 bispecific antibodies demonstrate increased colocalisationwith the lysosomal marker LAMP1, when compared with their monospecificcounterparts. These results indicate that once internalized, HER2×HER2bispecific antibodies are efficiently sorted towards lysosomalcompartments, making them suitable for a bispecific antibody drugconjugate approach.

Example 45 Inhibition of Proliferation of AU565 Cells Upon Incubationwith HER2 Monospecific or HER2×HER2 Bispecific Antibodies

HER2 bispecific antibodies were tested for their ability to inhibitproliferation of AU565 cells in vitro. Due to the high HER2 expressionlevels on AU565 cells (˜1.000.000 copies per cell as determined withQifi-kit), HER2 is constitutively active in these cells and thus notdependent on ligand-induced heterodimerization. In a 96-wells tissueculture plate (Greiner bio-one, Frickenhausen, Germany), 9.000 AU565cells were seeded per well in the presence of 10 μg/mL HER2 antibody orHER2×HER2 bispecific antibodies in serum-free cell culture medium. As acontrol, cells were seeded in serum-free medium without antibody orbispecific antibodies. After three days, the amount of viable cells wasquantified with Alamarblue (BioSource International, San Francisco, US)according to the manufacturer's instructions. Fluorescence was monitoredusing the EnVision 2101 Multilabel reader (PerkinElmer, Turku, Finland)with standard Alamarblue settings. The Alamarblue signal ofantibody-treated cells was plotted as a percentage relative to untreatedcells.

FIG. 47 depicts the fluorescent intensity of Alamarblue of AU565 cellsafter incubation with HER2 antibodies and HER2×HER2 bispecificantibodies. Herceptin® (trastuzumab) was included as positive controland demonstrated inhibition of proliferation as described by Juntilla TT. et al., Cancer Cell 2009; 15: 429-440. All HER2×HER2 bispecificantibodies were able to inhibit proliferation of AU565 cells. Bispecificantibodies: IgG1-005-ITL×IgG1-169-K409R and IgG1-025-ITL×IgG1-005-K409Rwere more effective compared to their monospecific antibody counterpartsin this assay.

Example 46 In Vitro and In Vivo Analysis of FcRn Binding by BispecificIgG1 Antibodies and Hinge-Deleted IgG1 Bispecific Antibodies ContainingOne or Two FcRn Binding Sites in the Fc Region

The present example illustrates the generation of asymmetricalbispecific molecules, i.e. molecules with different characteristics ineach Fab-arm according to the invention.

The neonatal Fc receptor (FcRn) is responsible for the long plasmahalf-life of IgG by protecting IgG from degradation. Afterinternalization of the antibody, FcRn binds to antibody Fc regions inendosomes, where the interaction is stable in the mildly acidicenvironment (pH 6.0). Upon recycling to the plasma membrane, where theenvironment is neutral (pH 7.4), the interaction is lost and theantibody is released back into the circulation. The Fc region of anantibody contains 2 FcRn binding sites, one in each heavy chain at theCH2-CH3 interfaces. An H435A mutation in the Fc region of the antibodyabrogates binding to FcRn (Shields, R. L., et al, J Biol Chem, 2001,Firan, M., et al, Int Immunol, 2001) and also the hinge region isthought to influence FcRn binding (Kim, J. K., et al., Mol Immunol.,1995). Furthermore, a role for bivalent over monovalent antibody bindingto FcRn has been suggested in efficient recycling (Kim, J. K., et al.,Scand J Immunol., 1994).

In this example the influence of FcRn binding valency is evaluated byasymmetric bispecific IgG1 molecules, containing a single FcRn bindingsite. The additional contribution of the hinge region is evaluated byasymmetric bispecific hinge-deleted IgG1 (Uni-G1) molecules.

FcRn binding of bispecific IgG1 or hinge-deleted IgG1 (Uni-G1) moleculescontaining no, 1 or 2 FcRn binding sites was measured by human and mouseFcRn ELISA. Antibodies IgG1-2F8-ITL, IgG1-7D8-K409R andIgG1-7D8-K409R-H435A monospecific molecules were produced as describedin example 2, 3, 4 and 5. Hinge-deleted IgG1 molecules Uni-G1-2F8-ITL,Uni-G1-7D8-K409R and Uni-G1-7D8-K409R-H435A monospecific molecules wereproduced as described in example 11. Bispecific IgG1 molecules weregenerated by 2-MEA induced Fab-arm exchange between IgG1-2F8-ITL andIgG1-7D8-K409R or IgG1-7D8-K409R-H435A molecules. Bispecifichinge-deleted IgG1 molecules were produced by incubation ofUni-G1-2F8-ITL with Uni-G1-7D8-K409R or Uni-G1-7D8-K409R-H435A. A 3-folddilution series of monospecific and bispecific IgG1 molecules andhinge-deleted IgG1 molecules were added to biotinylated human- ormouse-FcRn captured on a streptavidin-coated elisa plate followed byincubation at pH 6.0 and 7.4 for 1 hour. Bound antibody andhinge-deleted IgG1 molecules were visualized usinghorseradishperoxidase-labeled goat-anti-human (Fab)₂ as conjugate andABTS as substrate. Results were measured as optical density at awavelength of 405 nm using the EL808-Elisa-reader.

FIG. 48 shows the binding results of monovalent or bivalent IgG1antibodies and hinge-deleted IgG1 molecules to human FcRn (A) and mouseFcRn (B) at pH 6.0 and pH 7.4. As expected, all antibodies tested, both(bispecific) IgG1 and hinge-deleted IgG1 molecules, do not bindefficiently to FcRn (both human and mouse) at pH 7.4. At slightly acidiccondition (pH 6.0) monospecific IgG1-2F8-ITL and bispecific IgG1generated from IgG1-2F8-ITL and IgG1-7D8-K409R show bivalent bindingefficiencies to FcRn, albeit for mouse FcRn 3 fold higher compared tohuman, which mimics the positive control (IgG1-2F8) for FcRn binding.This indicates that the ITL mutation and the K409R do not disturbbinding to FcRn.

A clear effect of 2 vs 1 vs 0 FcRn interaction sites can be seen whenthe binding of the IgG1 molecules to human and mouse FcRn is compared atpH 6.0 (Figure XXA and B, pH6, left panel). IgG1-2F8-ITL, IgG1-7D8-K409Rand IgG1-2F8-ITL/IgG1-7D8-K409R (2 FcRn binding sites) bind comparableto the control (IgG1-2F8). The molecules with 0 FcRn binding sites,IgG1-7D8-K409R-H435A show no binding at all. The molecules with 1 FcRnbinding site, IgG1-2F8-ITL/IgG1-7D8-K409R-H435A, show intermediatebinding when compared to the molecules with 2 FcRn binding sites.

FIG. 48(A), pH 6.0, right panel shows the binding to human FcRn ofhinge-deleted IgG1 molecules (Uni-G1). All hinge-deleted molecules areimpaired in their interaction to human FcRn when compared to the controlIgG1 molecules (IgG1-2F8) indicating that the hinge is indeed ofinfluence in the interaction with FcRn when evaluated in an FcRn bindingELISA. No clear effect of 2 vs 1 vs 0 FcRn interaction sites can be seenwhen the binding to human FcRn at pH6.0 is compared of thesehinge-deleted molecules.

However, since the binding of human IgG to mouse FcRn is stronger, aclear effect of 2 vs 1 vs 0 FcRn interaction sites can be seen when thebinding of these hinge-deleted IgG molecules to mouse FcRn at pH 6.0 iscompared (FIG. 48(B), pH 6.0, right panel). The binding ofUni-G1-7D8-K409R-H435A/Uni-G1-2F8-ITL (1 FcRn binding site) isintermediate when compared to the binding of Uni-G1-2F8-ITL,Uni-G1-7D8-409R and Uni-G1-2F8-ITL/Uni-G1-7D8-K409R (2 FcRn bindingsites) and Uni-G1-2F8-ITL-H435A (0 FcRn binding sites, no binding).

Example 47 Her2×CD3 Bispecific Antibodies Tested in an In VitroCytotoxicity Assay

CD3 is a co-receptor in the T cell receptor complex expressed on matureT cells. Combination of a CD3 specific antibody Fab-arm with a tumorantigen specific antibody Fab-arm in a bispecific antibody would resultin the specific targeting of T cells to tumor cells, leading to T cellmediated tumor cell lysis. Likewise, CD3 positive T cells could betargeted to other derailed cells in the body, to infected cells ordirectly to pathogens.

Her2×CD3 bispecific antibodies were generated. Heavy and light chainvariable region sequences for the Her2 specific Fab-arm were asindicated for antibody 153 and 169 in Example 42. The following heavyand light chain variable region sequences for the CD3 specific Fab-armwere used:

YTH12.5 (Sequence as described by Routledge etal., Eur J Immunol. 1991, 21(11): 2717-25.) VHEVQLLESGGGLVQPGGSLRLSCAASGFTFSSFPMAWVRQAP YTH12.5GKGLEWVSTISTSGGRTYYRDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKFRQYSGGFDYWGQGTLVTVSS VLDIQLTQPNSVSTSLGSTVKLSCTLSSGNIENNYVHWYQLYE YTH12.5GRSPTTMIYDDDKRPDGVPDRFSGSIDRSSNSAFLTIHNVA IEDEAIYFCHSYVSSFNVFGGGTKLTVLhuCLB-T3/4 (Sequence as described by Parren etal., Res Immunol. 1991, 142(9): 749-63. Minoramino acid substitutions were introduced tomake the sequence resemble the closest human germline.) VHEVQLVESGGGLVKPGGSLRLSCAASGFTFSSYGMFWVRQAP huCLB-GKGLEWVATISRYSRYIYYPDSVKGRFTISRDNAKNSLYLQ T3/4MNSLRAEDTAVYYCARRPLYGSSPDYWGQGTLVTVSS VLEIVLTQSPATLSLSPGERATLSCSASSSVTYVHWYQQKPGQ huCLB-APRLLIYDTSKLASGIPARFSGSGSGTDFTLTISSLEPEDF T3/4 AVYYCFQGSGYPLTFGSGTKLEMR

All antibodies were expressed as IgG1,κ being modified in their Fcregions as described as follows: IgG1-Her2-153-K409R andIgG1-Her2-153-N297Q-K409R, IgG1-Her2-169-K409R, IgG1-hu-CLB-T3/4-F405Land IgG1-hu-CLB-T3/4-N297Q-F405L, IgG1-YTH12.5-F405L andIgG1-YTH12.5-N297Q-F405L.

Bispecific antibodies from these Her2 and CD3 specific antibodies weregenerated as described in Example 11 and tested in an in vitrocytotoxicity assay using AU565 cells.

AU565 cells were cultured to near confluency. Cells were washed twicewith PBS, and trypsinized for 5 minutes at 37° C. 12 mL culture mediumwas added to inactivate trypsin and cells were spun down for 5 min, 800rpm. Cells were resuspended in 10 mL culture medium and a single cellsuspension was made by passing the cells through a cellstrainer. 100 μLof a 5×10⁵ cells/mL suspension was added to each well of a 96-wellculture plate, and cells were incubated at least 3 hrs at 37° C., 5% CO2to allow adherence to the plate.

Peripheral blood mononuclear cells (PBMC) were isolated from blood fromhealthy volunteers using Leucosep 30 mL tubes, according to themanufacturer's protocol (Greiner Bio-one). T cells were isolated fromPBMC preparations by negative selection using the Untouched HumanT-cells Dynabead kit (Dynal). Isolated cells were resuspended in culturemedium to a final concentration op 7×10⁶ cells/mL.

Culture medium was removed from the adhered AU565 cells, and replacedwith 50 μl/well 2× concentrated antibody-dilution and 50 μl/well 7×10⁶ Tcells/mL (ratio effector:target=7:1). Plates were incubated for 3 daysat 37° C., 5% CO₂. Supernatants were removed and plates were washedtwice with PBS. To each well 150 μL culture medium and 15 μL Alamar bluewas added. Plates were incubated for 4 hours at 37° C., 5% CO₂, andabsorbance was measured (Envision, Perkin Elmer).

FIG. 49 shows that whereas control antibodies (Her2 monospecificIgG1-Herceptin, CD3 monospecific IgG1-YTH12.5 and monospecificIgG1-huCLB-T3/4, irrelevant antigen monospecific IgG1-b12, and CD3×b12bispecific antibodies) did not induce T cell mediated cytotoxicity,bispecific (Duo) Her2×CD3 antibodies huCLB/Her2-153, huCLB/Her2-169,YTH12.5/Her2-153 and YTH12.5/Her2-169 induced dose dependent T cellmediated cytotoxicity of AU565 cells. Bispecific antibodies containingHer2-169 were more potent than those containing Her2-153.

Mutants of IgG1-hu-CLB-T3/4, IgG1-YTH12.5 and Her2-153 were madecontaining a N297Q mutation to remove a glycosylation site;glycosylation at this site is critical for IgG-Fcgamma receptorinteractions (Bolt S et al., Eur J Immunol 1993, 23:403-411). FIG. 49shows that N297Q mutation and therefore absence of Fc glycosylation ofHer2×CD3 bispecific antibodies YTH12.5/Her2-153 and huCLB/Her2-153 didnot impact the potential to induce dose dependent T cell mediatedcytotoxicity of AU565 cells.

1. An in vitro method for generating a heterodimeric protein, saidmethod comprising the following steps: a) providing a first homodimericprotein comprising an Fc region of an immunoglobulin, said Fc regioncomprising a first CH3 region, b) providing a second homodimeric proteincomprising an Fc region of an immunoglobulin, said Fc region comprisinga second CH3 region, wherein the sequences of said first and second CH3regions are different and are such that the heterodimeric interactionbetween said first and second CH3 regions is stronger than each of thehomodimeric interactions of said first and second CH3 regions, c)incubating said first protein together with said second protein underreducing conditions sufficient to allow the cysteines in the hingeregion to undergo disulfide-bond isomerization, and d) obtaining saidheterodimeric protein.
 2. The in vitro method according to claim 1,wherein said first homodimeric protein and said second homodimericprotein are selected from the group consisting of (i) an Fc region, (ii)an antibody, (iii) a fusion protein comprising an Fc region, and (iv) aFc region conjugated to a prodrug, peptide, drug or a toxin.
 3. The invitro method according to claim 1, wherein said first and/or secondhomodimeric protein is a full-length antibody.
 4. (canceled)
 5. The invitro method according to claim 1, wherein said first and secondhomodimeric proteins are both antibodies and bind different epitopes. 6.The in vitro method according to claim 1, wherein the Fc region of thefirst homodimeric protein is of an isotype selected from the groupconsisting of IgG1, IgG2, IgG3 and IgG4 and wherein the Fc region of thesecond homodimeric protein is of an isotype selected from the groupconsisting of IgG1, IgG2, IgG3 and IgG4. 7-9. (canceled)
 10. The invitro method according to claim 1, wherein the heterodimeric interactionbetween said first and second proteins in the resulting heterodimericprotein is (a) such that no Fab-arm exchange can occur at 0.5 mM GSHunder the conditions described in Example 13, and/or (b) such that noFab-arm exchange occurs in vivo in mice under the conditions describedin Example
 14. 11. (canceled)
 12. The in vitro method according to claim1, wherein the heterodimeric interaction between said first and secondproteins in the resulting heterodimeric protein is more than two timesstronger, such as more than three times stronger, e.g. more than fivetimes stronger than the strongest of the two homodimeric interactions,e.g. when determined as described in Example
 30. 13. The in vitro methodaccording to claim 1, wherein the sequences of said first and second CH3regions are such that: (a) the dissociation constants of theheterodimeric interaction between said first and second proteins in theresulting heterodimeric protein is below 0.05 micromolar when assayed asdescribed in Example 30 and/or (b) the dissociation constants of bothhomodimeric interactions are above 0.01 micromolar, such as above 0.05micromolar, preferably between 0.01 and 10 micromolar, such as between0.05 and 10 micromolar, more preferably between 0.01 and 5, such asbetween 0.05 and 5 micromolar, even more preferably between 0.01 and 1micromolar, such as between 0.05 and 1 micromolar, between 0.01 and 0.5or between 0.01 and 0.1 micromolar when assayed as described in Example21.
 14. (canceled)
 15. The in vitro method according to claim 1, whereinthe sequences of said first and second CH3 regions contain amino acidsubstitutions at non-identical positions.
 16. (canceled)
 17. The invitro method according to claim 1, wherein said first homodimericprotein has no more than one amino acid substitution in the CH3 region,and the second homodimeric protein has no more than one amino acidsubstitution in the CH3 region relative to the wild-type CH3 regions.18. The in vitro method according to claim 1, wherein said firsthomodimeric protein has an amino acid substitution at a positionselected from the group consisting of: 366, 368, 370, 399, 405, 407 and409, and said second homodimeric protein has an amino acid substitutionat a position selected from the group consisting of: 366, 368, 370, 399,405, 407 and 409, and wherein said first homodimeric protein and saidsecond homodimeric protein is not substituted in the same positions. 19.The in vitro method according to claim 1, wherein (a) said firsthomodimeric protein has an amino acid other than Lys, Leu or Met atposition 409 and said second homodimeric protein has an amino acidsubstitution at a position selected from the group consisting of: 366,368, 370, 399, 405 and 407, (b) said first homodimeric protein has anamino acid other than Lys, Leu or Met at position 409 and said secondhomodimeric protein has an amino acid other than Phe at position 405,(c) said first homodimeric protein has an amino acid other than Lys, Leuor Met at position 409 and said second homodimeric protein has an aminoacid other than Phe, Arg or Gly at position 405, (d) said firsthomodimeric protein comprises a Phe at position 405 and an amino acidother than Lys, Leu or Met at position 409 and said second homodimericprotein comprises an amino acid other than Phe at position 405 and a Lysat position 409, (e) said first homodimeric protein comprises a Phe atposition 405 and an amino acid other than Lys, Leu or Met at position409 and said second homodimeric protein comprises an amino acid otherthan Phe, Arg or Gly at position 405 and a Lys at position 409, (f) saidfirst homodimeric protein comprises a Phe at position 405 and an aminoacid other than Lys, Leu or Met at position 409 and said secondhomodimeric protein comprises a Leu at position 405 and a Lys atposition 409, (g) said first homodimeric protein comprises a Phe atposition 405 and an Arg at position 409 and said second homodimericprotein comprises an amino acid other than Phe, Arg or Gly at position405 and a Lys at position 409, (h) said first homodimeric proteincomprises Phe at position 405 and an Arg at position 409 and said secondhomodimeric protein comprises a Leu at position 405 and a Lys atposition 409, (i) said first homodimeric protein comprises an amino acidother than Lys, Leu or Met at position 409 and said second homodimericprotein comprises a Lys at position 409, a Thr at position 370 and a Leuat position 405, (j) said first homodimeric protein comprises an Arg atposition 409 and said second homodimeric protein comprises a Lys atposition 409, a Thr at position 370 and a Leu at position 405, (k) saidfirst homodimeric protein comprises a Lys at position 370, a Phe atposition 405 and an Arg at position 409 and said second homodimericprotein comprises a Lys at position 409, a Thr at position 370 and a Leuat position 405, (l) said first homodimeric protein has an amino acidother than Lys, Leu or Met at position 409 and said second homodimericprotein has an amino acid other than Tyr, Asp, Glu, Phe, Lys, Gln, Arg,Ser or Thr at position 407, (m) said first homodimeric protein has anamino acid other than Lys, Leu or Met at position 409 and said secondhomodimeric protein has an Ala, Gly, His, Ile, Leu, Met, Asn, Val or Trpat position 407, (n) said first homodimeric protein has an amino acidother than Lys, Leu or Met at position 409 and said second homodimericprotein has a Gly, Leu, Met, Asn or Trp at position 407, (o) said firsthomodimeric protein has a Tyr at position 407 and an amino acid otherthan Lys, Leu or Met at position 409 and said second homodimeric proteinhas an amino acid other than Tyr, Asp, Glu, Phe, Lys, Gln, Arg, Ser orThr at position 407 and a Lys at position 409, (p) said firsthomodimeric protein has a Tyr at position 407 and an amino acid otherthan Lys, Leu or Met at position 409 and said second homodimeric proteinhas an Ala, Gly, His, Ile, Leu, Met, Asn, Val or Trp at position 407 anda Lys at position 409, (q) said first homodimeric protein has a Tyr atposition 407 and an amino acid other than Lys, Leu or Met at position409 and said second homodimeric protein has a Gly, Leu, Met, Asn or Trpat position 407 and a Lys at position 409, (r) said first homodimericprotein has a Tyr at position 407 and an Arg at position 409 and saidsecond homodimeric protein has an amino acid other than Tyr, Asp, Glu,Phe, Lys, Gln, Arg, Ser or Thr at position 407 and a Lys at position409, (s) said first homodimeric protein has a Tyr at position 407 and anArg at position 409 and said second homodimeric protein has an Ala, Gly,His, Ile, Leu, Met, Asn, Val or Trp at position 407 and a Lys atposition 409, (t) said first homodimeric protein has a Tyr at position407 and an Arg at position 409 and said second homodimeric protein has aGly, Leu, Met, Asn or Trp at position 407 and a Lys at position 409, (u)said first homodimeric protein has an amino acid other than Lys, Leu orMet at position 409, and the second homodimeric protein has (i) an aminoacid other than Phe, Leu and Met at position 368, or (ii) a Trp atposition 370, or (iii) an amino acid other than Asp, Cys, Pro, Glu orGln at position 399, (v) said first homodimeric protein has an Arg, Ala,His or Gly at position 409, and the second homodimeric protein has (i) aLys, Gln, Ala, Asp, Glu, Gly, His, Ile, Asn, Arg, Ser, Thr, Val, or Trpat position 368, or (ii) a Trp at position 370, or (iii) an Ala, Gly,Ile, Leu, Met, Asn, Ser, Thr, Trp, Phe, His, Lys, Arg or Tyr at position399, or (w) said first homodimeric protein has an Arg at position 409,and the second homodimeric protein has (i) an Asp, Glu, Gly, Asn, Arg,Ser, Thr, Val, or Trp at position 368, or (ii) a Trp at position 370, or(iii) a Phe, His, Lys, Arg or Tyr at position
 399. 20-42. (canceled) 43.The in vitro method according to claim 1, wherein (a) neither said firstnor said second homodimeric protein comprises a Cys-Pro-Ser-Cys sequencein the hinge region, or (b) both said first and said second homodimericprotein comprise a Cys-Pro-Pro-Cys sequence in the hinge region. 44.(canceled)
 45. The in vitro method according to claim 1, wherein saidfirst and second homodimeric proteins, except for any specifiedmutations, are human antibodies.
 46. The in vitro method according toclaim 1, wherein said first and second homodimeric proteins areheavy-chain antibodies.
 47. The in vitro method according to claim 1,wherein both said first and said second homodimeric proteins furthercomprise a light chain, wherein, optionally, said light chains aredifferent. 48-49. (canceled)
 50. The in vitro method according to claim1, wherein said first and second homodimeric proteins provided in stepa) and b) are purified.
 51. The in vitro method according to claim 1,wherein said first and/or second homodimeric protein is conjugated to adrug, a prodrug or a toxin or contains an acceptor group for the same.52-58. (canceled)
 59. The in vitro method according to claim 1, whereinthe reducing conditions in step c) comprise the addition of a reducingagent, e.g. a reducing agent selected from the group consisting of:2-mercaptoethylamine, dithiothreitol and tris(2-carboxyethyl)phosphineor chemical derivatives thereof.
 60. The in vitro method according toclaim 1, wherein step c) (i) is performed under reducing conditions witha redox potential between −150 and −600 mV, such as between −250 and−400 mV and/or (ii) comprises incubation for at least 90 min at atemperature of at least 20° C. in the presence of at least 25 mM2-mercaptoethylamine or in the presence of at least 0.5 mMdithiothreitol.
 61. (canceled)
 62. The in vitro method according toclaim 1, wherein step d) comprises removal of a reducing agent, e.g. bydesalting.
 63. A method for the selection of a bispecific antibodyhaving a desired property, said method comprising the steps of: a)providing a first set of homodimeric antibodies comprising antibodieswith different variable regions, wherein said antibodies of said firstset comprise identical first CH3 regions, b) providing a second set ofhomodimeric antibodies comprising antibodies with different variableregions or identical variable regions, wherein said antibodies of saidsecond set comprise identical second CH3 regions, wherein the sequencesof said first and second CH3 regions are different and are such that theheterodimeric interaction between said first and second CH3 regions isstronger than each of the homodimeric interactions of said first andsecond CH3 regions, c) incubating combinations of antibodies of saidfirst set and of said second set under reducing conditions sufficient toallow the cysteines in the hinge region to undergo disulfide-bondisomerization, thus generating a set of bispecific antibodies, d)optionally restoring the conditions to non-reducing, e) assaying theresulting set of bispecific antibodies for a given desired property, andf) selecting a bispecific antibody having the desired property. 64-65.(canceled)
 66. A method for producing a heterodimeric protein, saidmethod comprising the following steps: a) providing a first nucleic-acidconstruct encoding a first polypeptide comprising a first Fc region ofan immunoglobulin, said first Fc region comprising a first CH3 region,b) providing a second nucleic-acid construct encoding a secondpolypeptide comprising a second Fc region of an immunoglobulin, saidsecond Fc region comprising a first CH3 region, wherein the sequences ofsaid first and second CH3 regions are different and are such that theheterodimeric interaction between said first and second CH3 regions isstronger than each of the homodimeric interactions of said first andsecond CH3 regions, and wherein said first homodimeric protein has anamino acid other than Lys, Leu or Met at position 409 and said secondhomodimeric protein has an amino-acid substitution at a positionselected from the group consisting of: 366, 368, 370, 399, 405 and 407.and/or wherein the sequences of said first and second CH3 regions aresuch that the dissociation constants of homodimeric interactions of eachof the CH3 regions are between 0.01 and 10 micromolar, such as between0.05 and 10 micromolar, more preferably between 0.01 and 5, such asbetween 0.05 and 5 micromolar, even more preferably between 0.01 and 1micromolar, such as between 0.05 and 1 micromolar, between 0.01 and 0.5or between 0.01 and 0.1 when assayed as described in Example
 21. c)co-expressing said first and second nucleic-acid constructs in a hostcell, and d) obtaining said heterodimeric protein from the cell culture.67-70. (canceled)
 71. An expression vector comprising the nucleic-acidconstructs specified in claim
 66. 72. A host cell comprising thenucleic-acid constructs specified in claim
 66. 73. A heterodimericprotein obtained or obtainable by the method of claim
 1. 74. Aheterodimeric protein comprising a first polypeptide comprising a firstFc region of an immunoglobulin, said first Fc region comprising a firstCH3 region, and a second polypeptide comprising a second Fc region of animmunoglobulin, said second Fc region comprising a second CH3 region,wherein the sequences of said first and second CH3 regions are differentand are such that the heterodimeric interaction between said first andsecond CH3 regions is stronger than each of the homodimeric interactionsof said first and second CH3 regions, and wherein said first homodimericprotein has an amino acid other than Lys, Leu or Met at position 409 andsaid second homodimeric protein has an amino-acid substitution at aposition selected from the group consisting of: 366, 368, 370, 399, 405and 407 and/or wherein the sequences of said first and second CH3regions are such that the dissociation constants of homodimericinteractions of each of the CH3 regions are between 0.01 and 10micromolar, such as between 0.05 and 10 micromolar, more preferablybetween 0.01 and 5, such as between 0.05 and 5 micromolar, even morepreferably between 0.01 and 1 micromolar, such as between 0.05 and 1micromolar, between 0.01 and 0.5 or between 0.01 and 0.1 when assayed asdescribed in Example
 21. 75-80. (canceled)
 81. A pharmaceuticalcomposition comprising a heterodimeric protein according to claim 73 anda pharmaceutically-acceptable carrier.
 82. A method for inhibitinggrowth and/or proliferation and/or for killing of tumor cells comprisingadministration of a heterodimeric protein according to claim 73 to anindividual in need thereof.
 83. A pharmaceutical composition comprisinga heterodimeric protein according to claim 74 and apharmaceutically-acceptable carrier.
 84. A method for inhibiting growthand/or proliferation and/or for killing of tumor cells comprisingadministration of a heterodimeric protein according to claim 74 to anindividual in need thereof.